Ligands for metabotropic glutamate receptors and inhibitors of naaladase

ABSTRACT

The present invention relates to novel compounds and formulations thereof which compounds are ligands, e.g., agonists or antagonists, for a metabotropic glutamate receptor or a NAALADase enzyme or both. The present invention also relates to methods of modulating the activity of a metabotropic glutamate receptor or a NAALADase enzyme or both, e.g., in a subject in need thereof, using a compound or formulation of the present invention. The present invention also relates to methods of treating a subject suffering from a chronic or acute disease, malady or condition due at least in part to an abnormality in the activity of an endogenous metabotropic glutamate receptor or a NAALADase enzyme or both, using a compound or formulation of the present invention.

RELATED APPLICATIONS

This application is a continuation of copending U.S. patent applicationSer. No. 10/374,765, filed Feb. 25, 2003; which is a continuation ofU.S. patent application Ser. No. 09/662,767, filed Sep. 15, 2000, nowU.S. Pat. No. 6,528,499; which is a continuation-in-part of U.S. patentapplication Ser. No. 09/559,978, filed Apr. 27, 2000, now U.S. Pat. No.6,479,470; which claimed the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 60/188,031, filed Mar. 9, 2000; U.S.Provisional Patent Application Ser. No. 60/166,915, filed Nov. 22, 1999;and U.S. Provisional Patent Application Ser. No. 60/131,627, filed Apr.28, 1999, which applications are all incorporated herein by reference intheir entirety.

GOVERNMENT SUPPORT

This invention was made with support from the National Institutes ofHealth and Department of Defense; the government, therefore, has certainrights in the invention.

BACKGROUND OF THE INVENTION

Glutamate is a major excitatory neurotransmitter in the mammaliancentral nervous system. The neurotransmitter activity of glutamate isprimarily mediated by ligand-gated ion channels. The observation thatglutamate also induces responses mediated by second messengers has ledto the discovery of a distinct group of glutamate receptors coupled to Gproteins, termed metabotropic receptors (mGluRs). Schoepp and Conn,Trends Pharmacol. Sci. 14: 13-20 (1993). The first described action ofthe glutamate metabotropic receptors was inositol phospholipid (PI)hydrolysis. Nicoletti et al., J. Neurochem. 46: 40-46 (1986) andSugiyama et al., Nature 325: 531-533 (1987). Molecular cloningtechniques have revealed a large family of metabotropic receptors withdistinct transduction mechanisms, patterns of expression andsensitivities to glutamate agonists. Schoepp and Conn, supra.

Consistent with the molecular heterogeneity observed for themetabotropic receptors, electrophysiological studies have suggesteddiverse roles for these receptors in synaptic plasticity, presynapticinhibition and regulation of cell excitability by ion channelmodulation. Bashir et al., Nature 363: 347-363 (1993); Linden et al.,Neuron 7: 81-89 (1991); Baskys and Malenka, J. Physiol. (Lond.) 444:687-701 (1991); Charpak et al. Nature 347: 765-767 (1990); and Lesterand Jahr, Neuron 5: 741-749 (1990). However, the specific mGluRreceptors mediating these cellular functions are largely undefined.

Evidence for a physiological role for specific mGluR subtypes has beenderived from work with selective agonists and antagonists of thereceptors. For example, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylicacid (ACPD) is a selective and potent activator of the mGluR1, mGluR2,mGluR3 and mGluR5 receptors. Masu et al., Nature 349: 760-765 (1991);Abe et al., J. Biol. Chem. 267: 13361-13368 (1992); Tanabe et al.,Neuron 8: 169-179 (1992); and Tanabe et al., J. Neurosci. 13: 1372-1378(1993). L-2-amino-4-phosphonobutryic acid (L-AP4) has been shown toactivate mGluR4 and mGluR6. Id., Thomsen et al., Eur. J. Pharmacol. 227:361-362 (1992); Nakajima et al., J. Biol. Chem. 268:11868-11873 (1993).L-AP4 inhibits transmitter release and voltage-dependent calcium entryin selected brain and spinal cord neurons. Koerner and Cotman, BrainRes. 216: 192-198 (1981); Trombley and Westbrook, J. Neurosci.12:2-43-2050 (1992); and Sahara and Westbrook, J. Neurosci. 13:3041-3050 (1993). But in retinal bipolar neurons, postsynaptic L-AP4receptors activate aphosphodiesterase. Nawy and Jahr, Nature 346:269-271 (1990).

Multiple mGluR subtypes can be present within the same group of neurons.As the cellular and subcellular localization of specific mGluRs may beimportant in shaping incoming sensory information, it is important toidentify other receptors of the mGluR group. Once identified, specificagonists and antagonists can be prepared to modulate the responsesassociated with the receptor. Quite surprisingly, the present inventionidentifies a L-AP4 sensitive receptor that modulates transmitter releasein neurons that express neither mGluR4 nor mGluR6, and fulfills otherrelated needs.

As alluded to above, the metabotropic glutamate receptors (mGluRs) are aheterogeneous family of G-protein linked receptors that are coupled tomultiple second messenger systems. These include the negative modulationof adenylate cyclase, activation of phosphoinositide-specificphospholipase C, and modulation of ion channel currents [Science, 1992,258, 597; Trends in Pharmacol. Sci. 1993, 14, 13; J. Med. Chem. 1995,1417]. Three types of mGluR receptors have been identified. The group Ireceptors couple to phosphoinositide hydrolysis and include mGluR₁ andmGluR₅; group II receptors are coupled to the inhibition of cyclicadenosine 5′-monophosphate(cAMP) formation and include mGluR₂ andmGluR₃. Group III receptors (mGluR₄, mGluR₆, mGluR₇ and mGluR₈) alsocouple negatively to cAMP. Each of the mGluR subtypes is thusdistinguished on the basis of its pharmacology and sequence homology.Excessive activation of glutamate receptors or disturbances in thecellular mechanisms that protect against the potential adverseconsequences of physiological glutamate receptor activation has beenimplicated in the pathogenesis of a diverse number of neurologicaldisorders. These disorders include epilepsy, ischemia, central nervoussystem trauma, neuropathic pain, and chronic neurodegenerative diseases.Because of the ubiquitous distribution of glutamatergic synapses, mGluRshave the potential to participate in a wide variety of functions in theCNS. In addition, because of the wide diversity and heterogeneousdistribution of the mGluRs subtypes, the opportunity exists fordeveloping highly selective drugs that affect a limited number of CNSfunctions. The mGluRs therefore provide novel targets for thedevelopment of therapeutic agents that could have a dramatic impact ontreatment of a wide variety of psychiatric and neurological disorders.

Ischemia, a localized tissue anemia resulting from the obstruction ofthe inflow of arterial blood, can cause extensive damage when it occursin the brain or central nervous system. Central nervous tissue, and to alesser extent peripheral nervous tissue, has poor reparative abilities.Thus damage to nervous tissue causes significant permanent disabilityand is a frequent cause of death. Damage to nervous tissue may occur inmany ways, not only through ischemia in cerebrovascular accidents, butalso in cerebral circulatory disturbances, episodes of absolute andrelative hypoxia, from metabolic disturbances and from various forms oftrauma.

Global ischemia occurs under conditions in which blood flow to theentire brain ceases for a period of time, such as may result fromcardiac arrest. Focal ischemia occurs under conditions in which aportion of the brain is deprived of its normal blood supply, such as mayresult from thromboembolytic occlusion of a cerebral vessel, traumatichead injury, edema, and brain tumors. In areas of focal ischemia ordamage, there is a core of more profound damage, surrounded by aperifocal penumbra of lesser damage. This is because the neurons in thepenumbra can for a time maintain homeostasis thus rendering thempotentially more salvageable by pharmacological agents.

Both global and focal ischemic conditions have the potential forproducing widespread neuronal damage, even if the ischemic condition istransient. Although some permanent neuronal injury may occur in theinitial mixture following cessation of blood flow to the brain, thedamage in global and focal ischemia occurs over hours or even daysfollowing the ischemic onset. Much of this neuronal damage is attributedto glutamate toxicity and secondary consequences of reperfusion of thetissue, such as the release of vasoactive products by damagedendothelium, and the release by the damaged tissues of cytotoxicproducts including free radicals, leukotrienes, and the like.

Glutamate neurotoxicity, which is a major factor in ischemic neuronalinjury, appears to begin with resumption of oxidative metabolism andthus occurs both during reversible ischemia and during recovery. Manyattempts have been made to avoid this problem by blocking of the variousreceptors including NMDA receptors, AMPA receptors, Kainate receptors,and MGR receptors, which are stimulated by glutamate and are alsostrongly involved in nerve cell death occurring after the onset ofglobal or focal ischemia. When ischemia occurs, such as during a strokeor heart attack, there is an excessive release of endogenous glutamate,resulting in the overstimulation of NMDA receptors, AMPA receptors,Kainate receptors, and MGR receptors. Interaction of the glutamate withthese receptors causes the ion channel associated with these receptorsto open, allowing a flow of cations across the cell membrane. This fluxof ions, particularly Ca²⁺ into the cells, plays an important role innerve cell death.

Prostate cancer is now the leading form of cancer among men and thesecond most frequent cause of death from cancer in men. It is estimatedthat more than 165,000 new cases of prostate cancer were diagnosed in1993, and more than 35,000 men died from prostate cancer in that year.Additionally, the incidence of prostate cancer has increased by 50%since 1981, and mortality from this disease has continued to increase.Previously, most men died of other illnesses or diseases before dyingfrom their prostate cancer. We now face increasing morbidity fromprostate cancer as men live longer and the disease has the opportunityto progress. Current therapies for prostate cancer focus exclusivelyupon reducing levels of dihydrotestosterone to decrease or preventgrowth of prostate cancer. In addition to the use of digital rectalexamination and transrectal ultrasonography, prostate-specific antigen(PSA) concentration is frequently used in the diagnosis of prostatecancer.

PSA is a protein produced by prostate cells and is frequently present atelevated levels in the blood of men who have prostate cancer. PSA hasbeen shown to correlate with tumor burden, serve as an indicator ofmetastatic involvement, and provide a parameter for following theresponse to surgery, irradiation, and androgen replacement therapy inprostate cancer patients. It should be noted that Prostate SpecificAntigen (PSA) is a completely different protein from Prostate SpecificMembrane Antigen (PSMA). The two proteins have different structures andfunctions and should not be confused because of their similarnomenclature.

In 1993, the molecular cloning of a prostate-specific membrane antigen(PSMA) was reported as a potential prostate carcinoma marker andhypothesized to serve as a target for imaging and cytotoxic treatmentmodalities for prostate cancer. Antibodies against PSMA have beendescribed and examined clinically for diagnosis and treatment ofprostate cancer. In particular, Indium-111 labelled PSMA antibodies havebeen described and examined for diagnosis of prostate cancer anditrium-labelled PSMA antibodies have been described and examined for thetreatment of prostate cancer.

PSMA is expressed in prostatic ductal epithelium and is present inseminal plasma, prostatic fluid and urine. In 1996, it was found thatthe expression of PSMA cDNA actually confers the activity of NAALADase.This is entirely unexpected because until recently NAALADase researchhas been limited to its role in the brain and its effect onneurotransmitters whereas PSMA has been described and examined for thediagnosis and therapy of prostate cancer.

The dipeptide NAAG is an abundant nervous system specific peptide whichis present in synaptic vesicles and released upon neuronal stimulationin several systems. As a major peptidic component of the brain, NAAG ispresent in levels comparable to that of the major inhibitoryneurotransmitter gamma-aminobutyric acid (GADA). Although NAAG was firstisolated in 1964, there was little activity toward elucidating its rolein the CNS until the deleterious nature of excess glutamate in a varietyof disease states became apparent. Due to its structural similarity toglutamate, NAAG has been suggested to have a variety of roles similar tothose of glutamate itself, including functioning as a neurotransmitteror a cotransmitter, neuromodulator, or as a precursor of theneurotransmitter glutamate. NAAG has elicited excitatory responses bothin vitro and in vivo, but is significantly less potent than glutamate.

In 1988, a brain enzyme, NAALADase, was identified which hydrolyzes NAAGto N-acetylaspartate (NAA) and glutamate. NAALADase, which derives itsname from its structural specificity for N-acetylated acidic dipeptides,is a membrane-bound metallopeptidase having a denatured molecular massof 94 kDa[x], that catabolizes NAAG to N-acetylaspartate (NAA) andglutamate. It has been demonstrated that [³H]NAAG is degraded in vivo byan enzyme with the pharmacological characteristics of NAALADase, whichsupports a role for NAALADase in the metabolism of endogenous NAAG.

Rat NAALADase activity has been extensively characterized anddemonstrates a high affinity for hydrolysis of its putative substrateNAAG, with a Km=140 nM. Recently, NAALADase also has been shown tocleave the non-acetylated peptide, aspartylglutamate, with highaffinity. Research has also found that the enzyme is membrane-bound,stimulated by chloride ions, and inhibited by polyvalent cationchelators, suggesting that it is a metallopeptidase.

In animals, NAALADase is enriched in synaptic plasma membranes and isprimarily localized to neural tissue and the kidneys. NAALADase has notbeen found in large quantities in the mammalian liver, heart, pancreas,or spleen. Examination of NAAG and NAALADase has been conducted forseveral different human and animal pathological conditions. It has beendemonstrated that intra-hippocampal injections of NAAG elicit prolongedseizure activity. More recently, it was reported that rats geneticallyprone to epileptic seizures have a persistent increase in their basallevel of NAALADase activity. These observations are consistent with thehypothesis that increased availability of synaptic glutamate elevatesseizure susceptibility, and suggest that NAALADase inhibitors mayprovide anti-epileptic activity.

NAAG and NAALADase have also been implicated in the pathogenesis of ALSand in the pathologically similar animal disease called HereditaryCanine Spinal Muscular Atrophy (HCSMA). It has been shown thatconcentrations of NAAG and its metabolites—NAA, glutamate andaspartate—are elevated two- to three-fold in the cerebrospinal fluid ofALS patients and HCSMA dogs.

In addition, NAALADase activity is significantly increased (two- tothree-fold) in post-mortem spinal cord tissue from ALS patients andHCSMA dogs. Although highly speculative, NAALADase inhibitors may beclinically useful in curbing the progression of ALS if increasedmetabolism of NAAG is responsible for the alterations of CSF levels ofthese acidic amino acids and peptides. Abnormalities in NAAG levels andNAALADase activity have also been documented in post-mortemschizophrenic brain, specifically in the prefrontal and limbicbrainregions, underscoring the importance of examining the metabolism of NAAGin the pathophysiology of schizophrenia. The identification andpurification of NAALADase led to the proposal of another role for NAAG:specifically that the dipeptide may serve as a storage form of synapticglutamate.

Only a few NAALADase inhibitors have been identified and those that havebeen identified have only been used in non-clinical neurologicalresearch. Examples of such inhibitors include general metallopeptidaseinhibitors such as o-phenanthrolene, metal chelators such as EGTA andEDTA, and peptide analogs such as quisqualic acid and beta-NAAG.

SUMMARY OF THE INVENTION

Certain embodiments of the present invention relate to new ligands formetabotropic glutamate receptors and compositions comprising theligands. The pharmaceutical compositions may be used to influenceglutamate receptor-controlled cells, including neurons and glial cellsin the central nervous system.

In additional embodiments, the present invention consists of inhibitorsof NAALADase enzyme activity and compositions comprising them.Additional embodiments of the present invention consist of methods oftreatment of glutamate abnormalities and associated nervous tissueinsult in a animal by inhibition of NAALADase enzyme with theaforementioned inhibitors or compositions thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts certain preferred embodiments of the compounds of thepresent invention.

FIG. 2 depicts the effects of six compounds of the present invention onthe activity of NAALADase.

FIG. 3 depicts the effect of a compound of the present invention at sixsubtypes of metabotropic glutamate receptors (mGluRs).

FIG. 4 depicts the effect of a compound of the present invention at asingle subtype of metabotropic glutamate receptor.

FIG. 5 depicts the effects of a compound of the present invention and2-(phosphomethyl)pentanedioic acid (PMPA) on the activity of NAAGpeptidase in rat brain membranes.

FIG. 6 depicts the effects of a compound of the present invention, PMPA,and Quis on the activity of rat NAAG peptidase.

FIG. 7 depicts the effects of five compounds of the present invention onthe activity of rat NAAG peptidase.

FIG. 8 depicts the effects of a compound of the present invention, PMPA,and Quis on the activity of prostate specific membrane antigen (PMSA).

FIG. 9 depicts the effects of two compounds of the present invention onthe activity of rat NAAG peptidase.

FIG. 10 depicts certain compounds of the present invention, theiractivity against NAAG peptidase, and their activity against certainmetabotropic glutamate receptors.

FIG. 11 depicts certain compounds of the present invention, theiractivity against NAAG peptidase, and their activity against certainmetabotropic glutamate receptors.

FIG. 12 depicts certain compounds of the present invention, theiractivity against NAAG peptidase, and their activity against certainmetabotropic glutamate receptors.

FIG. 13 depicts at low magnification the antiangiogenic effects of acompound of the present invention on a glioblastoma xenograft.

FIG. 14 depicts at high magnification the antiangiogenic effects of acompound of the present invention on a glioblastoma xenograft.

FIG. 15 presents a plot of tumor volume versus duration of treatment forglioblastoma xenografts treated with a compound of the present inventionat 10 and 100 μM, contrasted with a control.

FIG. 16 depicts the synthesis of 4-(R)-benzyl-FN11.

FIG. 17 depicts the synthesis of 4-(R)-methyl-FN11.

FIG. 18 depicts the synthesis of 4-(S)-methyl-FN11.

FIG. 19 depicts the synthesis of 2-(S)-methyl-FN11.

FIG. 20 depicts the synthesis of a γ-tetrazole analogue.

FIG. 21 depicts the synthesis of an α-tetrazole analogue.

FIG. 22 depicts the synthesis of 2-(S)-benzyl-FN11.

FIG. 23 depicts the synthesis of a dimer of LY-354740.

FIG. 24 depicts the synthesis of an optically pure dimer of LY-354740.

FIG. 25 depicts the design scheme for the glutamate dimer, FN-11.

FIG. 26 depicts the peptidase activity of various FN-11-based ureas.

FIG. 27 depicts the design scheme for urea-based NAALADase inhibitors.

FIG. 28 depicts the activities of certain ureas derived from LY-354740.

FIG. 29 presents the neuroprotective action of ABHxD-I,2-aminobicyclo[2.1.1]hexane-2,5-dicarboxylic acid, in three models ofNMDA toxicity which shows a lack of effect in cultures of pure neuronswithout glia (A), while significant protection is seen in the modelusing mixed cortical neuronal-glia culture (B) and when neurons arerescued with medium from glial cells exposed to the protective compound(C). Control refers to cells treated only with NMDA (without theprotective compound). ABHxD was used at 10 μM concentration. Barsrepresent means and SEM from 16-30 measurements.

FIG. 30 shows the effects of FN compounds at 300 μM on NMDA toxicity inmixed cultures of cortical neuronal-glial cells (model B). Controlrefers to cells treated only with NMDA (without the protectivecompound). ABHxD-1 (10 μM) was used as a positive control. Barsrepresent means and SEM from 8-40 measurements.

FIG. 31 shows the effects of FN6 enantiomers at 100 μM on NMDA toxicityin mixed cultures of cortical neuronal-glial cells (model B). Controlrefers to cells treated only with NMDA (without the protectivecompound). ABHxD-1 (10 μM) was used as a positive control. Barsrepresent means and SEM from 8-11 measurements.

FIG. 32 shows the effects of FN6 enantiomers at 100 μM on NMDA toxicityin cortical neuronal cells exposed to medium from drug-treated glialcells (model C). Control refers to cells treated only with NMDA (andmedium from untreated glia). ABHxD-1 (10 μM) was used as positivecontrol. Results with the Guilford compound PMPA are shown forcomparison. Bars represent means and SEM from 12-30 measurements.

FIG. 33 shows the effects of FN compounds at 100 μM on NMDA toxicity incortical neuronal cells exposed to medium from drug-treated glial cells(model C). Control refers cells treated only with NMDA (and medium fromuntreated glia). ABHxD-1 (10 μM) was used as a positive control. Barsrepresent means and SEM from 8-61 measurements.

FIG. 34 depicts the protective effects of novel ureas against neuronalcell death induced by NMDA in primary cultures of mouse corticalneurons. Cultures of cortical neurons were treated for 10 min with 20 μMNMDA, followed by treatment with culture medium collected from culturesof cortical astrocytes pretreated for 24 hours with the indicatedcompounds at 1 μM concentration. Neuronal viability was assessed 24hours later by measurements of LDH accumulation. Results are expressedas a percent of cell death induced by NMDA alone. Bars represent meanvalues s.e.m. from 16-32 determinations.

DETAILED DESCRIPTION OF THE INVENTION

To date, all of the commonly used agonist and antagonists employed inbiological studies of the mGluRs are amino acids, often embodying astructurally rigidified glutamate-like core [Neuropharmacology, 36, 1-11(1997); Neuropharmacology 37, 1-12 (1998); Neuropharmacology 35,1661-1672 (1996); J. Med. Chem. 38, 1417 (1995); J. Med. Chem. 41, 347(1998); Current Pharmaceutical Design, 1, 355 (1995)] During our effortsto identify potent and selective ligands acting at these receptors, wehave discovered mGluR₃ selective agonists that contains only acidgroups.

Our studies began from the dipeptide N-acetyl-L-aspartate-L-glutamate(NAAG), a dipeptide that is quite abundant in the brain, and which isbelieved to act as a transmitter or co-transmitter in the centralnervous system, much like glutamate itself. NAAG exhibits excitatoryproperties both in vitro and in vivo, but it is less active thanglutamate and represents a storage form of glutamate. In studies usingcell lines transfected with mGluR₁₋₆, NAAG was found to selectivelyactivate the mGluR₃ receptor with an EC₅₀ value in the range of 65±20 μM[J. Neurochem. 69, 174 (1997)]. Recently, a brain enzyme was identifiedthat is specific for the cleavage of N-acetylated α-linked acidicdipeptides, and as a result this enzyme was named NAALADase [J. Biol.Chem., 1987, 262, 14498].

NAAG and NAALADase have been implicated in several pathologicalconditions relating to glutamate abnormalities and neurotoxicity. Forexample, it has been demonstrated that intra-hippocampal injections ofNAAG elicit prolonged seizure activity [Proc. Natl. Acad. Sci. USA, 80(1983), 1116-1119]. It has also been reported that rats geneticallyprone to epileptic seizures demonstrate a persistent increase in theirbasal levels of NAALADase activity. [Brain Research, 593 (1992),140-143]. These results lend support to the hypothesis that theincreased availability of synaptic glutamate elevates seizuresusceptibility. As a consequence, it has been suggested that NAALADaseinhibitors may provide effective anti-epileptic therapies.

NAAG and NAALADase have also been implicated in the pathogenesis of ALS(Amyotrophic Lateral Sclerosis) [Brain Research, 556 (1991), 151-156].As such, NAALADase inhibitors might be clinically useful in curbing theprogression of ALS if an increased metabolism of NAAG is responsible foralterations in the CSF levels of these acidic amino acids [see Ann.Neurol. 28 (1990), 18-25].

Certain phosphonate analogs of NAAG, such as2-(phosphonomethyl)-pentanedioic acid, have been reported to act aspotent inhibitors of NAALADase [J. Med. Chem. 39, 619 (1996)].Interestingly, while this compound was reported to show little in theway receptor activity, we found that it does in fact act as an agonistat mGluR₃.

Compounds of the Invention

Based upon this unexpected result, we were led to explore the activityof related analogs. As part of our design strategy, we decided toexplore the activity of NAAG-like analogs that were missing the amidebond present between the Asp and Glu residue, (the standardketomethylene type substitution), but also from which the N-acetyl groupwas deleted, as this particular group was reported not to be an absoluterequirement for NAALADase activity.

Accordingly, a series of compounds was prepared and studied for mGluRactivity. Of the compounds synthesized, the compound comprised of anacetone moiety flanked by the two pentanedioic acid groups (A) proved tobe interesting, as it retained significant mGluR₃ activity.

Based on this observation, we also chose to explore the activity ofcompounds in which the central carbonyl group of A was replaced byP(O)OH, CHOH, O, S, SO, SO₂, and R₃CHOH with the idea that this compoundmight act not only as an mGluR₃ selective ligand, but that it might alsofunction as a NAALADase inhibitor. The phosphorous compound (B) wasparticularly potent as a NAALADase inhibitor, with an IC₅₀ of 4 nM.Additional data on the new compounds are provided herein.

In certain embodiments, the compounds of the present invention arerepresented by structure 1:

wherein

X is selected from the group consisting of —C(O)—, —C(S)—, —P(O)(OR)—,—S(O)₂—, —C(R)(OR)—, and —C(R)(SR)—;

Y is selected, independently for each occurrence, from the groupconsisting of (CR₂)_(n), (NR)_(n), and a bond;

Z is selected, independently for each occurrence, from the groupconsisting of C(R), C(NR₂), and C(NHacyl);

W is selected, independently for each occurrence, from the groupconsisting of (CR₂)_(m), (NR)_(m), and a bond;

G is selected, independently for each occurrence, from the groupconsisting of H, —COOH, —SO₃H, —P(O)(OH)₂, —SR, and 2-R-tetrazol-5-yl;

R is selected, independently for each occurrence, from the groupconsisting of H, alkyl, heteroalkyl, aryl, heteroaryl, and aralkyl; andalso including a negative charge for instances of R bonded to aheteroatom;

m and n are integers selected, independently for each occurrence, fromthe range 0 to 3 inclusive; and

the stereochemical configuration at any stereocenter of a compoundrepresented by 1 is R, S, or a mixture of these configurations.

In certain embodiments, the compounds of the present invention arerepresented by structure 1 and the attendant definitions, wherein X is—C(O)—.

In certain embodiments, the compounds of the present invention arerepresented by structure 1 and the attendant definitions, wherein Y isindependently for each occurrence (NR)_(n).

In certain embodiments, the compounds of the present invention arerepresented by structure 1 and the attendant definitions, wherein Z isindependently for each occurrence C(R).

In certain embodiments, the compounds of the present invention arerepresented by structure 1 and the attendant definitions, wherein W isindependently for each occurrence (CR₂)_(m).

In certain embodiments, the compounds of the present invention arerepresented by structure 1 and the attendant definitions, wherein G isselected, independently for each occurrence, from the group consistingof H, —COOH, —SR, and 2-R-tetrazol-5-yl.

In certain embodiments, the compounds of the present invention arerepresented by structure 1 and the attendant definitions, wherein m andn are integers selected, independently for each occurrence, from 1 and2.

In certain embodiments, the compounds of the present invention arerepresented by structure 1 and the attendant definitions, wherein X is—C(O)—; and Y is independently for each occurrence (NR)_(n).

In certain embodiments, the compounds of the present invention arerepresented by structure 1 and the attendant definitions, wherein X is—C(O)—; and Z is independently for each occurrence C(R).

In certain embodiments, the compounds of the present invention arerepresented by structure 1 and the attendant definitions, wherein X is—C(O)—; and W is independently for each occurrence (CR₂)_(m).

In certain embodiments, the compounds of the present invention arerepresented by structure 1 and the attendant definitions, wherein X is—C(O)—; and G is selected, independently for each occurrence, from thegroup consisting of H, —COOH, —SR, and 2-R-tetrazol-5-yl.

In certain embodiments, the compounds of the present invention arerepresented by structure 1 and the attendant definitions, wherein X is—C(O)—; Y is independently for each occurrence (NR)_(n); and Z isindependently for each occurrence C(R).

In certain embodiments, the compounds of the present invention arerepresented by structure 1 and the attendant definitions, wherein X is—C(O)—; Y is independently for each occurrence (NR)_(n); and W isindependently for each occurrence (CR₂)_(m).

In certain embodiments, the compounds of the present invention arerepresented by structure 1 and the attendant definitions, wherein X is—C(O)—; Y is independently for each occurrence (NR)_(n); and G isselected, independently for each occurrence, from the group consistingof H, —COOH, —SR, and 2-R-tetrazol-5-yl.

In certain embodiments, the compounds of the present invention arerepresented by structure 1 and the attendant definitions, wherein X is—C(O)—; Y is independently for each occurrence (NR)_(n); Z isindependently for each occurrence C(R); and W is independently for eachoccurrence (CR₂)_(m).

In certain embodiments, the compounds of the present invention arerepresented by structure 1 and the attendant definitions, wherein X is—C(O)—; Y is independently for each occurrence (NR)_(n); W isindependently for each occurrence (CR₂)_(m); and G is selected,independently for each occurrence, from the group consisting of H,—COOH, —SR, and 2-R-tetrazol-5-yl.

In certain embodiments, the compounds of the present invention arerepresented by structure 1 and the attendant definitions, wherein X is—C(O)—; Y is independently for each occurrence (NR)_(n); Z isindependently for each occurrence C(R); W is independently for eachoccurrence (CR₂)_(m); and G is selected, independently for eachoccurrence, from the group consisting of H, —COOH, —SR, and2-R-tetrazol-5-yl.

In certain embodiments, the compounds of the present invention arerepresented by structure 2:

wherein

X is selected from the group consisting of —C(O)—, —C(S)—, —P(O)(OR)—,—S(O)₂—, —C(R)(OR)—, and —C(R)(SR)—;

Y is selected, independently for each occurrence, from the groupconsisting of (CR₂)_(n), (NR)_(n), and a bond;

G is selected, independently for each occurrence, from the groupconsisting of H, —COOH, —SO₃H, —P(O)(OH)₂, and 2-R-tetrazol-5-yl;

R is selected, independently for each occurrence, from the groupconsisting of H, alkyl, heteroalkyl, aryl, heteroaryl, and aralkyl; andalso including a negative charge for instances of R bonded to aheteroatom;

n is an integer selected, independently for each occurrence, from therange 0 to 3 inclusive; and

the stereochemical configuration at any stereocenter of a compoundrepresented by 2 is R, S, or a mixture of these configurations.

In certain embodiments, the compounds of the present invention arerepresented by structure 2 and the attendant definitions, wherein X is—C(O)—.

In certain embodiments, the compounds of the present invention arerepresented by structure 2 and the attendant definitions, wherein Y isindependently for each occurrence (NR)_(n).

In certain embodiments, the compounds of the present invention arerepresented by structure 2 and the attendant definitions, wherein G isselected, independently for each occurrence, from the group consistingof —COOH, —SO₃H, —P(O)(OH)₂, and 2-R-tetrazol-5-yl.

In certain embodiments, the compounds of the present invention arerepresented by structure 2 and the attendant definitions, wherein G isselected, independently for each occurrence, from the group consistingof —COOH, and 2-R-tetrazol-5-yl.

In certain embodiments, the compounds of the present invention arerepresented by structure 2 and the attendant definitions, wherein X is—C(O)—; and Y is independently for each occurrence (NR)_(n).

In certain embodiments, the compounds of the present invention arerepresented by structure 2 and the attendant definitions, wherein X is—C(O)—; Y is independently for each occurrence (NR)_(n); and G isselected, independently for each occurrence, from the group consistingof —COOH, —SO₃H, —P(O)(OH)₂, and 2-R-tetrazol-5-yl.

In certain embodiments, the compounds of the present invention arerepresented by structure 2 and the attendant definitions, wherein X is—C(O)—; Y is independently for each occurrence (NR)_(n); and G isselected, independently for each occurrence, from the group consistingof —COOH, and 2-R-tetrazol-5-yl.

In certain embodiments, a compound of the present invention isrepresented by structure 1 or 2 and the attendant definitions, whereinthe compound is a single stereoisomer.

In certain embodiments, a compound of the present invention isrepresented by structure 1 or 2 and the attendant definitions, whereinthe compound is a ligand for a metabotropic glutamate receptor.

In certain embodiments, a compound of the present invention isrepresented by structure 1 or 2 and the attendant definitions, whereinthe compound is an agonist of a metabotropic glutamate receptor.

In certain embodiments, a compound of the present invention isrepresented by structure 1 or 2 and the attendant definitions, whereinthe compound is an antagonist of a metabotropic glutamate receptor.

In certain embodiments, a compound of the present invention isrepresented by structure 1 or 2 and the attendant definitions, whereinthe compound is a ligand for a single subtype of metabotropic glutamatereceptor.

In certain embodiments, a compound of the present invention isrepresented by structure 1 or 2 and the attendant definitions, whereinthe compound is an agonist of a single subtype of metabotropic glutamatereceptor.

In certain embodiments, a compound of the present invention isrepresented by structure 1 or 2 and the attendant definitions, whereinthe compound is an antagonist of a single subtype of metabotropicglutamate receptor.

In certain embodiments, a compound of the present invention isrepresented by structure 1 or 2 and the attendant definitions, whereinthe compound is an inhibitor of NAALADase.

In certain embodiments, the present invention relates to apharmaceutical composition, comprising a compound represented bystructure 1 or 2 and the attendant definitions; and pharmaceuticallyacceptable excipient.

In certain embodiments, the present invention relates to a method ofinhibiting NAALADase in a mammal, comprising the step of administeringto a mammal a therapeutically effective amount of a compound representedby structure 1 or 2 and the attendant definitions.

In certain embodiments, the present invention relates to a method ofagonising a metabotropic glutamate receptor in a mammal, comprising thestep of administering to a subject mammal a therapeutically effectiveamount of a compound represented by structure 1 or 2 and the attendantdefinitions.

In certain embodiments, the present invention relates to a method ofantagonising a metabotropic glutamate receptor in a mammal, comprisingthe step of administering to a subject mammal a therapeuticallyeffective amount of a compound represented by structure 1 or 2 and theattendant definitions.

In certain embodiments, the present invention relates to a method ofagonising a single subtype of metabotropic glutamate receptor in amammal, comprising the step of administering to a mammal atherapeutically effective amount of a compound represented by structure1 or 2 and the attendant definitions.

In certain embodiments, the present invention relates to a method ofantagonising a single subtype of metabotropic glutamate receptor in amammal, comprising the step of administering to a mammal atherapeutically effective amount of a compound represented by structure1 or 2 and the attendant definitions.

Another aspect of the present invention relates to methods of treatingischemia, in particular global and focal ischemia, using compositionswhich inhibit N-Acetylated alpha-Linked Acidic Dipeptidase (NAALADase)enzyme activity in humans and warm-blooded animals. Certain compoundsrepresented by structure 1 or 2 and the attendant definitions areinhibitors of NAALADase. Those of ordinary skill in the art will be ableto ascertain using no more than routine experimentation which compoundsof the present invention are antagonists of NAALADase.

NAALADase is an enzyme which is a membrane-bound metalloprotease thathydrolyzes the dipeptide, N-acetyl-L-aspartate-L-glutamate (NAAG) toyield glutamate and N-acetylaspartate. The methods of the presentinvention include using compositions containing phosphinic acidderivatives that inhibit NAALADase enzyme activity and which have beenfound useful for the treatment of ischemia. The amino acid L-glutamateis a neurotransmitter that mediates fast neuronal excitation in amajority of synapses in the central nervous system (CNS). Once releasedinto the synapse, L-glutamate can stimulate the N-methyl-D-aspartate(NMDA) receptor, a subtype of an excitatory amino acid receptor. It hasbeen discovered that excessive activation of the NMDA receptor has beenimplicated in a variety of acute as well as chronic neuropathologicalprocesses such as ischemia, epilepsy and Huntington's disease. Thus,considerable effort has focused on finding novel therapeutic agents toantagonize the postsynaptic effects of L-glutamate medicated through theNMDA receptor.

Certain embodiments of the present invention consist of a method fortreating ischemia which comprises the step of administering to an animalsuffering from an ischemia a NAALADase inhibitor and pharmaceuticallyacceptable carrier for said NAALADase inhibitor. In methods of treatingstroke, particularly acute ischemic stroke, and global ischemia causedby drowning, head trauma and so forth, a NAALADase inhibitor can beco-administered with one or more agents active in reducing the risk ofstroke, such as aspirin or ticlopidine (preferably ticlopidine, whichhas been demonstrated to reduce the risk of a second ischemic event).Co-administration can be in the form of a single formulation (combining,for example, a NAALADase inhibitor and ticlopidine with pharmaceuticallyacceptable excipients, optionally segregating the two active ingredientsin different excipient mixtures designed to independently control theirrespective release rates and durations) or by independent administrationof separate formulations containing the active agents.

If desired, the pharmaceutical composition to be administered may alsocontain minor amounts of non-toxic auxiliary substances such as wettingor emulsifying agents, pH buffering agents and the like, such as forexample, sodium acetate, sorbitan monolaurate, triethanolamine oleate,etc.

The NAALADase inhibitors of this invention are generally administered asa pharmaceutical composition which comprises a pharmaceutical excipientin combination with the inhibitor. The level of the drug in aformulation can vary within the full range employed by those skilled inthe art, namely, from about 0.01 percent weight (% w) to about 99.99% wof the drug based on the total formulation and about 0.01% w to 99.99% wexcipient. Preferably, the formulation will be about 3.5 to 60% byweight of the NAALADase inhibitor, with the rest being suitablepharmaceutically excipients.

DEFINITIONS

For convenience, before further description of the present invention,certain terms employed in the specification, examples, and appendedclaims are collected here.

“NAALADase” as used herein refers to N-Acetylated Alpha-Linked AcidicDipeptidase. The enzyme was originally named for it's substratespecificity for hydrolyzing N-acetylated alpha-linked acidic dipeptides.Currently, it is known that the enzyme has a broader range of substratespecificity than originally discovered, particularly that the enzymedoes not require N-acetylation or alpha-linkage. Thus, as used herein“NAALADase” encompasses other names used in the literature such as NAAGhydrolyzing enzyme and NAALA dipeptidase.

The term “inhibition”, in the context of enzyme inhibition, relates toreversible enzyme inhibition such as competitive, uncompetitive, andnoncompetitive inhibition. This can be experimentally distinguished bythe effects of the inhibitor on the reaction kinetics of the enzyme,which may be analyzed in terms of the basic Michaelis-Menten rateequation. Competitive inhibition occurs when the inhibitor can combinewith the free enzyme in such a way that it competes with the normalsubstrate for binding at the active site. A competitive inhibitor reactsreversibly with the enzyme to form an enzyme-inhibitor complex [EI].Following the Michaelis-Menten formalism, we can define the inhibitorconstant, K[i], as the dissociation constant of the enzyme-inhibitorcomplex. Thus, in accordance with the above and as used herein, K[i] isessentially a measurement of affinity between a molecule, and itsreceptor, or in relation to the present invention, between the presentinventive compounds and the enzyme to be inhibited. It should be notedthat IC50 is a related term used when defining the concentration oramount of a compound which is required to cause a 50% inhibition of thetarget enzyme.

The term “ischemia” relates to localized tissue anemia due toobstruction of the inflow of arterial blood. Global ischemia occursunder conditions in which blood flow to the entire brain ceases for aperiod of time, such as may result from cardiac arrest. Focal ischemiaoccurs under conditions in which a portion of the brain is deprived ofits normal blood supply, such as may result from thromboembolyticocclusion of a cerebral vessel, traumatic head injury, edema, and braintumors.

The term “nervous tissue” refers to the various components that make upthe nervous system including neurons, neural support cells, glia,Schwann cells, vasculature contained within and supplying thesestructures, the central nervous system, the brain, the brain stem, thespinal cord, the junction of the central nervous system with theperipheral nervous system, the peripheral nervous system and alliedstructures.

The term “nervous function” refers to the various functions of thenervous system and its parts which are manifest in sensing theenvironment, awareness of it, homeostasis to it and interaction with itas shown, by example, in the ability to perform activities of dailyliving, work, cogitation and speech.

The term “nervous insults” refers to damage to nervous tissue whichincludes brain and nervous tissue damage and destruction, in whole or inpart, and resultant morbidity, disability, neurologic deficia and death.Nervous insult can be from various origins including ischemia, hypoxia,cerebrovascular accident, metabolic, toxic, neurotoxic, trauma, surgery,iatrogenic, pressure, mass effect, hemorrhage, thermal, chemical,radiation, vasospasm, neurodegenerative disease, neurodegenerativeprocess, infection, Parkinson's disease, amyotrophic lateral sclerosis,myelination/demyelination processes, epilepsy, cognitive disorders,glutamate abnormalities, and their secondary effects.

The term “glutamate abnormalities” refers to any condition, disease, ordisorder that involves glutamate, and includes but is not limited to thenervous insults listed above.

The term “glutamate modulator” refers to any composition of matter,alone or in combination with another agent, which affects the level ofglutamate in an animal, including a human being.

The term “neuroprotective” is an effect which reduces, arrests, orameliorates nervous insult and is protective, resuscitative orrevivative for nervous tissue that has suffered nervous insult.

The term “treatment” refers to any process, action, application,therapy, or the like, wherein an animal, including a human being, issubject to medical aid with the object of improving the animal'scondition, directly or indirectly. The method of this invention fortreating global ischemia comprises administering internally to a subjectexpected to be benefited thereby with an effective amount of a NAALADaseinhibitor. Doses of this isomer included in the present methods andpharmaceutical compositions are an efficacious, nontoxic quantity.Persons skilled in the art using routine clinical testing are able todetermine optimum doses. The desired dose is administered to a subjectfrom 1 to 6 or more times daily, orally, rectally, parenterally, ortopically and may follow a higher initial amount administered as a bolusdose.

The term “nucleophile” is recognized in the art, and as used hereinmeans a chemical moiety having a reactive pair of electrons.

The term “electrophile” is art-recognized and refers to chemicalmoieties which can accept a pair of electrons from a nucleophile asdefined above. Electrophilic moieties useful in the method of thepresent invention include halides and sulfonates.

The term “electron-withdrawing group” is recognized in the art, anddenotes the tendency of a substituent to attract valence electrons fromneighboring atoms, i.e., the substituent is electronegative with respectto neighboring atoms. A quantification of the level ofelectron-withdrawing capability is given by the Hammett sigma (s)constant. This well known constant is described in many references, forinstance, J. March, Advanced Organic Chemistry, McGraw Hill BookCompany, New York, (1977 edition) pp. 251-259. The Hammett constantvalues are generally negative for electron donating groups (s[P]=−0.66for NH₂) and positive for electron withdrawing groups (s[P]=0.78 for anitro group), s[P] indicating para substitution. Exemplaryelectron-withdrawing groups include nitro, ketone, aldehyde, sulfonyl,trifluoromethyl, —CN, chloride, and the like. Exemplaryelectron-donating groups include amino, methoxy, and the like.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In certain embodiments, a straightchain or branched chain alkyl has 30 or fewer carbon atoms in itsbackbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ for branched chain),and more preferably 20 or fewer. Likewise, preferred cycloalkyls havefrom 3-10 carbon atoms in their ring structure, and more preferably have5, 6 or 7 carbons in the ring structure.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Preferred alkyl groups are lower alkyls. Incertain embodiments, a substituent designated herein as alkyl is a loweralkyl.

The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido,ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromaticmoieties, —CF₃, —CN, or the like. The term “aryl” also includespolycyclic ring systems having two or more cyclic rings in which two ormore carbons are common to two adjoining rings (the rings are “fusedrings”) wherein at least one of the rings is aromatic, e.g., the othercyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, arylsand/or heterocyclyls.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl,phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

The terms ortho, meta and para apply to 1,2-, 1,3- and 1,4-disubstitutedbenzenes, respectively. For example, the names 1,2-dimethylbenzene andortho-dimethylbenzene are synonymous.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromaticmoiety, —CF₃, —CN, or the like.

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen,sulfur and phosphorous.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The terms “amine” and “amino” are art recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀ and R′₁₀ each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R₈, or R₉ and R₁₀ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R₈ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In preferred embodiments, only one of R₉ or R₁₀can be a carbonyl, e.g., R₉, R₁₀ and the nitrogen together do not forman imide. In even more preferred embodiments, R₉ and R₁₀ (and optionallyR′₁₀) each independently represent a hydrogen, an alkyl, an alkenyl, or—(CH₂)_(m)—R₈. Thus, the term “alkylamine” as used herein means an aminegroup, as defined above, having a substituted or unsubstituted alkylattached thereto, i.e., at least one of R₉ and R₁₀ is an alkyl group.

The term “acylamino” is art-recognized and refers to a moiety that canbe represented by the general formula:

wherein R₉ is as defined above, and R′₁₁ represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉, R₁₀ are as defined above. Certain embodiments of the amidewill not include imides which may be unstable.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In certain embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R₈, wherein m and R₈ are defined above.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

The term “carbonyl” is art recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₈ or apharmaceutically acceptable salt, R′₁₁ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above. WhereX is an oxygen and R₁₁ or R′₁₁ is not hydrogen, the formula representsan “ester”. Where X is an oxygen, and R₁₁ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR₁₁ is a hydrogen, the formula represents a “carboxylic acid”. Where Xis an oxygen, and R′₁₁ is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiolcarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thiolester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiolcarboxylic acid.” Where X is a sulfur and R₁₁′ ishydrogen, the formula represents a “thiolformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH₂)_(m)—R₈,where m and R₈ are described above.

The term “sulfonate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is as defined above.

The term “sulfonamido” is art recognized and includes a moiety that canbe represented by the general formula:

in which R₉ and R′₁₁ are as defined above.

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₉ and R₁₀ are as defined above.

The terms “sulfoxido” or “sulfinyl”, as used herein, refers to a moietythat can be represented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

A “phosphoryl” can in general be represented by the formula:

wherein Q₁ represented S or O, and R₄₆ represents hydrogen, a loweralkyl or an aryl. When used to substitute, e.g., an alkyl, thephosphoryl group of the phosphorylalkyl can be represented by thegeneral formula:

wherein Q₁ represented S or O, and each R₄₆ independently representshydrogen, a lower alkyl or an aryl, Q₂ represents O, S or N. When Q₁ isan S, the phosphoryl moiety is a “phosphorothioate”.

A “phosphoramidite” can be represented in the general formula:

wherein R₉ and R₁₀ are as defined above, and Q₂ represents O, S or N.

A “phosphonamidite” can be represented in the general formula:

wherein R₉ and R₁₀ are as defined above, Q₂ represents O, S or N, andR₄₈ represents a lower alkyl or an aryl, Q₂ represents O, S or N.

A “selenoalkyl” refers to an alkyl group having a substituted selenogroup attached thereto. Exemplary “selenoethers” which may besubstituted on the alkyl are selected from one of —Se-alkyl,—Se-alkenyl, —Se-alkynyl, and —Se—(CH₂)_(m)—R₈, m and R₈ being definedabove.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

The phrase “protecting group” as used herein means temporarymodifications of a potentially reactive functional group which protectit from undesired chemical transformations. Examples of such protectinggroups include esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991).

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described hereinabove. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalencies of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

A “polar solvent” means a solvent which has a dipole moment (e) of 2.9or greater, such as DMF, THF, ethylene glycol dimethyl ether, DMSO,acetone, acetonitrile, methanol, ethanol, isopropanol, n-propanol,t-butanol or 2-methoxyethyl ether. Preferred solvents are DMF, diglyme,and acetonitrile.

An “aprotic solvent” means a solvent that is not a hydrogen bond donor.Examples of such solvents are acetonitrile, toluene, DMF, diglyme, THFor DMSO.

A “polar, aprotic solvent” means a solvent which has a dipole moment (ε)of 2.9, and is not a hydrogen bond donor, for example DMF, acetonitrile,DMSO and THF.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Alsofor purposes of this invention, the term “hydrocarbon” is contemplatedto include all permissible compounds having at least one hydrogen andone carbon atom. In a broad aspect, the permissible hydrocarbons includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic organic compounds which can besubstituted or unsubstituted.

Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the compounds described above, formulatedtogether with one or more pharmaceutically acceptable carriers(additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscularor intravenous injection as, for example, a sterile solution orsuspension; (3) topical application, for example, as a cream, ointmentor spray applied to the skin; or (4) intravaginally or intrarectally,for example, as a pessary, cream or foam.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect in at least a sub-population of cells in an animal ata reasonable benefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

As set out above, certain embodiments of the present compounds maycontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable acids. The term “pharmaceutically-acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ during the final isolation andpurification of the compounds of the invention, or by separatelyreacting a purified compound of the invention in its free base form witha suitable organic or inorganic acid, and isolating the salt thusformed. Representative salts include the hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm.Sci. 66:1-19)

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. The term “pharmaceutically-acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ during the final isolation and purification of thecompounds, or by separately reacting the purified compound in its freeacid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically-acceptable metal cation, with ammonia,or with a pharmaceutically-acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like. (See, forexample, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Administration of the adenosine antagonists and agonists for use in themethod of this invention can be via any of the accepted modes ofadministration. These methods include, but are not limited to, oral,parenteral, transdermal, intraarticular and otherwise systemicadministration. Oral administration is preferred. The compounds areadministered in a therapeutically effective amount either alone or incombination with a suitable pharmaceutically acceptable carrier orexcipient.

Depending on the intended mode of administration, the adenosineantagonist or agonist of choice may be incorporated in anypharmaceutically acceptable dosage form, such as, for example, tablets,transdermal patches, pills, capsules, powders, liquids, suspensions,emulsions, aerosols or the like, preferably in unit dosage formssuitable for single administration of precise dosages, or sustainedrelease dosage forms for continuous controlled administration.Preferably the dosage form will include a pharmaceutically acceptableexcipient and, in addition, may contain other medicinal agents,pharmaceutical agents, carriers, adjuvants, and the like.

For solid dosage forms, non-toxic carriers include but are not limitedto, for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, the polyalkylene glycols, talcum,cellulose, glucose, sucrose and magnesium carbonate. Liquidpharmaceutically administrable dosage forms can, for example, comprise asolution or suspension of an active adenosine agent and optionalpharmaceutical adjuvants in a carrier, such as, for example, water,saline aqueous dextrose, glycerol, ethanol and the like, to thereby forma solution or suspension. If desired, the pharmaceutical composition tobe administered may also contain minor amounts of non-toxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like. Typical examples of such auxiliary agents are sodiumacetate, sorbitan monolaurate, triethanolamine, sodium acetate,triethanolamine oleate, etc. Actual methods of preparing such dosageforms are known, or will be apparent, to those skilled in the art; forexample, see: Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa., 16th Edition, 1980. The composition of theformulation to be administered will, in any event, contain a quantity ofthe active adenosine agent in an amount effective for treatment.

Parenteral administration is generally characterized by injection,either subcutaneously, intramuscularly or intravenously. Injectables canbe prepared in conventional forms, either as liquid solutions orsuspension, solid forms suitable for solution or suspension in liquidprior to injection, or as emulsions. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol and the like. Inaddition, if desired, the injectable pharmaceutical compositions to beadministered may also contain minor amounts of non-toxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like.

The amount of active adenosine antagonist or agonist administered will,of course, be dependent on the subject being treated, the severity andnature of the affliction, the manner of administration, the potency andpharmacodynamics of the particular agent and the judgement of theprescribing physician. However, the therapeutically effective dosage foruse in this invention will generally be in the range from about 0.01 mug/kg (body weight) to 5 mg/kg.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about1 percent to about ninety-nine percent of active ingredient, preferablyfrom about 5 percent to about 70 percent, most preferably from about 10percent to about 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically-acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the compoundin a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject compounds may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given in formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administrations are preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracistemally and topically, as by powders, ointments ordrops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisinvention for a patient, when used for the indicated analgesic effects,will range from about 0.0001 to about 100 mg per kilogram of body weightper day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition).

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the subject compounds, as described above,formulated together with one or more pharmaceutically acceptablecarriers (additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscularor intravenous injection as, for example, a sterile solution orsuspension; (3) topical application, for example, as a cream, ointmentor spray applied to the skin; or (4) intravaginally or intravectally,for example, as a pessary, cream or foam.

The compounds according to the invention may be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other pharmaceuticals.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable carriers and can also beadministered in conjunction with antimicrobial agents such aspenicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy, thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticaleffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as “Applied Animal Nutrition”,W.H. Freedman and CO., San Francisco, U.S.A., 1969 or “Livestock Feedsand Feeding” 0 and B books, Corvallis, Ore., U.S.A., 1977).

Combinatorial Libraries

In the current era of drug development, high throughput screening ofthousands to millions of compounds plays a key role. High throughputscreening generally incorporates automation and robotics to enabletesting these thousands to millions of compounds in one or morebioassays in a relatively short period of time. This high capacityscreening technique requires enormous amounts of “raw materials” havingimmense molecular diversity to fill available capacity. Accordingly,combinatorial chemistry will play a significant role in meeting thisdemand for new molecules for screening. Once “leads” are identifiedusing high throughput screening techniques, combinatorial chemistry willbe advantageously used to optimize these initial leads (whichanalogs/variants will be tested in the same high throughput screeningassay(s) that identified the initial lead).

A combinatorial library for the purposes of the present invention is amixture of chemically-related compounds which may be screened togetherfor a desired property; said libraries may be in solution or covalentlylinked to a solid support. The preparation of many related compounds ina single reaction greatly reduces and simplifies the number of screeningprocesses which need to be carried out. Screening for the appropriatebiological, pharmaceutical, agrochemical or physical property may bedone by conventional methods.

Diversity in a library can be created at a variety of different levels.For instance, the substrate aryl groups used in a combinatorial approachcan be diverse in terms of the core aryl moiety, e.g., a variegation interms of the ring structure, and/or can be varied with respect to theother substituents.

A variety of techniques are available in the art for generatingcombinatorial libraries of small organic molecules. See, for example,Blondelle et al. (1995) Trends Anal. Chem. 14:83; the Affymax U.S. Pat.Nos. 5,359,115 and 5,362,899: the Ellman U.S. Pat. No. 5,288,514: theStill et al. PCT publication WO 94/08051; Chen et al. (1994) JACS116:2661: Kerr et al. (1993) JACS 115:252; PCT publications WO92/10092,WO93/09668 and WO91/07087; and the Lerner et al. PCT publicationWO93/20242). Accordingly, a variety of libraries on the order of about16 to 1,000,000 or more diversomers can be synthesized and screened fora particular activity or property.

In an exemplary embodiment, a library of substituted diversomers can besynthesized using the subject reactions adapted to the techniquesdescribed in the Still et al. PCT publication WO 94/08051, e.g., beinglinked to a polymer bead by a hydrolyzable or photolyzable group, e.g.,located at one of the positions of substrate. According to the Still etal. technique, the library is synthesized on a set of beads, each beadincluding a set of tags identifying the particular diversomer on thatbead. In one embodiment, which is particularly suitable for discoveringenzyme inhibitors, the beads can be dispersed on the surface of apermeable membrane, and the diversomers released from the beads by lysisof the bead linker. The diversomer from each bead will diffuse acrossthe membrane to an assay zone, where it will interact with an enzymeassay. Detailed descriptions of a number of combinatorial methodologiesare provided below.

A) Direct Characterization

A growing trend in the field of combinatorial chemistry is to exploitthe sensitivity of techniques such as mass spectrometry (MS), e.g.,which can be used to characterize sub-femtomolar amounts of a compound,and to directly determine the chemical constitution of a compoundselected from a combinatorial library. For instance, where the libraryis provided on an insoluble support matrix, discrete populations ofcompounds can be first released from the support and characterized byMS. In other embodiments, as part of the MS sample preparationtechnique, such MS techniques as MALDI can be used to release a compoundfrom the matrix, particularly where a labile bond is used originally totether the compound to the matrix. For instance, a bead selected from alibrary can be irradiated in a MALDI step in order to release thediversomer from the matrix, and ionize the diversomer for MS analysis.

B) Multipin Synthesis

The libraries of the subject method can take the multipin libraryformat. Briefly, Geysen and co-workers (Geysen et al. (1984) PNAS81:3998-4002) introduced a method for generating compound libraries by aparallel synthesis on polyacrylic acid-grated polyethylene pins arrayedin the microtitre plate format. The Geysen technique can be used tosynthesize and screen thousands of compounds per week using the multipinmethod, and the tethered compounds may be reused in many assays.Appropriate linker moieties can also been appended to the pins so thatthe compounds may be cleaved from the supports after synthesis forassessment of purity and further evaluation (c.f., Bray et al. (1990)Tetrahedron Lett 31:5811-5814; Valerio et al. (1991) Anal Biochem197:168-177; Bray et al. (1991) Tetrahedron Lett 32:6163-6166).

C) Divide-Couple-Recombine

In yet another embodiment, a variegated library of compounds can beprovided on a set of beads utilizing the strategy ofdivide-couple-recombine (see, e.g., Houghten (1985) PNAS 82:5131-5135;and U.S. Pat. Nos. 4,631,211; 5,440,016; 5,480,971). Briefly, as thename implies, at each synthesis step where degeneracy is introduced intothe library, the beads are divided into separate groups equal to thenumber of different substituents to be added at a particular position inthe library, the different substituents coupled in separate reactions,and the beads recombined into one pool for the next iteration.

In one embodiment, the divide-couple-recombine strategy can be carriedout using an analogous approach to the so-called “tea bag” method firstdeveloped by Houghten, where compound synthesis occurs on resin sealedinside porous polypropylene bags (Houghten et al. (1986) PNAS82:5131-5135). Substituents are coupled to the compound-bearing resinsby placing the bags in appropriate reaction solutions, while all commonsteps such as resin washing and deprotection are performedsimultaneously in one reaction vessel. At the end of the synthesis, eachbag contains a single compound.

D) Combinatorial Libraries by Light-Directed, Spatially AddressableParallel Chemical Synthesis

A scheme of combinatorial synthesis in which the identity of a compoundis given by its locations on a synthesis substrate is termed aspatially-addressable synthesis. In one embodiment, the combinatorialprocess is carried out by controlling the addition of a chemical reagentto specific locations on a solid support (Dower et al. (1991) Annu RepMed Chem 26:271-280; Fodor, S. P. A. (1991) Science 251:767; Pirrung etal. (1992) U.S. Pat. No. 5,143,854; Jacobs et al. (1994) TrendsBiotechnol 12:19-26). The spatial resolution of photolithography affordsminiaturization. This technique can be carried out through the useprotection/deprotection reactions with photolabile protecting groups.

The key points of this technology are illustrated in Gallop et al.(1994) J Med Chem 37:1233-1251. A synthesis substrate is prepared forcoupling through the covalent attachment of photolabilenitroveratryloxycarbonyl (NVOC) protected amino linkers or otherphotolabile linkers. Light is used to selectively activate a specifiedregion of the synthesis support for coupling. Removal of the photolabileprotecting groups by light (deprotection) results in activation ofselected areas. After activation, the first of a set of amino acidanalogs, each bearing a photolabile protecting group on the aminoterminus, is exposed to the entire surface. Coupling only occurs inregions that were addressed by light in the preceding step. The reactionis stopped, the plates washed, and the substrate is again illuminatedthrough a second mask, activating a different region for reaction with asecond protected building block. The pattern of masks and the sequenceof reactants define the products and their locations. Since this processutilizes photolithography techniques, the number of compounds that canbe synthesized is limited only by the number of synthesis sites that canbe addressed with appropriate resolution. The position of each compoundis precisely known; hence, its interactions with other molecules can bedirectly assessed.

In a light-directed chemical synthesis, the products depend on thepattern of illumination and on the order of addition of reactants. Byvarying the lithographic patterns, many different sets of test compoundscan be synthesized simultaneously; this characteristic leads to thegeneration of many different masking strategies.

E) Encoded Combinatorial Libraries

In yet another embodiment, the subject method utilizes a compoundlibrary provided with an encoded tagging system. A recent improvement inthe identification of active compounds from combinatorial librariesemploys chemical indexing systems using tags that uniquely encode thereaction steps a given bead has undergone and, by inference, thestructure it carries. Conceptually, this approach mimics phage displaylibraries, where activity derives from expressed peptides, but thestructures of the active peptides are deduced from the correspondinggenomic DNA sequence. The first encoding of synthetic combinatoriallibraries employed DNA as the code. A variety of other forms of encodinghave been reported, including encoding with sequenceable bio-oligomers(e.g., oligonucleotides and peptides), and binary encoding withadditional non-sequenceable tags.

1) Tagging with Sequenceable Bio-Oligomers

The principle of using oligonucleotides to encode combinatorialsynthetic libraries was described in 1992 (Brenner et al. (1992) PNAS89:5381-5383), and an example of such a library appeared the followingyear (Needles et al. (1993) PNAS 90:10700-10704). A combinatoriallibrary of nominally 7⁷ (=823,543) peptides composed of all combinationsof Arg, Gln, Phe, Lys, Val, D-Val and Thr (three-letter amino acidcode), each of which was encoded by a specific dinucleotide (TA, TC, CT,AT, TT, CA and AC, respectively), was prepared by a series ofalternating rounds of peptide and oligonucleotide synthesis on solidsupport. In this work, the amine linking functionality on the bead wasspecifically differentiated toward peptide or oligonucleotide synthesisby simultaneously preincubating the beads with reagents that generateprotected OH groups for oligonucleotide synthesis and protected NH₂groups for peptide synthesis (here, in a ratio of 1:20). When complete,the tags each consisted of 69-mers, 14 units of which carried the code.The bead-bound library was incubated with a fluorescently labeledantibody, and beads containing bound antibody that fluoresced stronglywere harvested by fluorescence-activated cell sorting (FACS). The DNAtags were amplified by PCR and sequenced, and the predicted peptideswere synthesized. Following such techniques, compound libraries can bederived for use in the subject method, where the oligonucleotidesequence of the tag identifies the sequential combinatorial reactionsthat a particular bead underwent, and therefore provides the identity ofthe compound on the bead.

The use of oligonucleotide tags permits exquisitely sensitive taganalysis. Even so, the method requires careful choice of orthogonal setsof protecting groups required for alternating co-synthesis of the tagand the library member. Furthermore, the chemical lability of the tag,particularly the phosphate and sugar anomeric linkages, may limit thechoice of reagents and conditions that can be employed for the synthesisof non-oligomeric libraries. In certain embodiments, the librariesemploy linkers permitting selective detachment of the test compoundlibrary member for assay.

Peptides have also been employed as tagging molecules for combinatoriallibraries. Two exemplary approaches are described in the art, both ofwhich employ branched linkers to solid phase upon which coding andligand strands are alternately elaborated. In the first approach (Kerr JM et al. (1993) J Am Chem Soc 115:2529-2531), orthogonality in synthesisis achieved by employing acid-labile protection for the coding strandand base-labile protection for the compound strand.

In an alternative approach (Nikolaiev et al. (1993) Pept Res 6:161-170),branched linkers are employed so that the coding unit and the testcompound can both be attached to the same functional group on the resin.In one embodiment, a cleavable linker can be placed between the branchpoint and the bead so that cleavage releases a molecule containing bothcode and the compound (Ptek et al. (1991) Tetrahedron Lett32:3891-3894). In another embodiment, the cleavable linker can be placedso that the test compound can be selectively separated from the bead,leaving the code behind. This last construct is particularly valuablebecause it permits screening of the test compound without potentialinterference of the coding groups. Examples in the art of independentcleavage and sequencing of peptide library members and theircorresponding tags has confirmed that the tags can accurately predictthe peptide structure.

2) Non-sequenceable Tagging: Binary Encoding

An alternative form of encoding the test compound library employs a setof non-sequenceable electrophoric tagging molecules that are used as abinary code (Ohlmeyer et al. (1993) PNAS 90:10922-10926). Exemplary tagsare haloaromatic alkyl ethers that are detectable as theirtrimethylsilyl ethers at less than femtomolar levels by electron capturegas chromatography (ECGC). Variations in the length of the alkyl chain,as well as the nature and position of the aromatic halide substituents,permit the synthesis of at least 40 such tags, which in principle canencode 2⁴⁰ (e.g., upwards of 10¹²) different molecules. In the originalreport (Ohlmeyer et al., supra) the tags were bound to about 1% of theavailable amine groups of a peptide library via a photocleavableo-nitrobenzyl linker. This approach is convenient when preparingcombinatorial libraries of peptide-like or other amine-containingmolecules. A more versatile system has, however, been developed thatpermits encoding of essentially any combinatorial library. Here, thecompound would be attached to the solid support via the photocleavablelinker and the tag is attached through a catechol ether linker viacarbene insertion into the bead matrix (Nestler et al. (1994) J. Org.Chem. 59:4723-4724). This orthogonal attachment strategy permits theselective detachment of library members for assay in solution andsubsequent decoding by ECGC after oxidative detachment of the tag sets.

Although several amide-linked libraries in the art employ binaryencoding with the electrophoric tags attached to amine groups, attachingthese tags directly to the bead matrix provides far greater versatilityin the structures that can be prepared in encoded combinatoriallibraries. Attached in this way, the tags and their linker are nearly asunreactive as the bead matrix itself. Two binary-encoded combinatoriallibraries have been reported where the electrophoric tags are attacheddirectly to the solid phase (Ohlmeyer et al. (1995) PNAS 92:6027-6031)and provide guidance for generating the subject compound library. Bothlibraries were constructed using an orthogonal attachment strategy inwhich the library member was linked to the solid support by aphotolabile linker and the tags were attached through a linker cleavableonly by vigorous oxidation. Because the library members can berepetitively partially photoeluted from the solid support, librarymembers can be utilized in multiple assays. Successive photoelution alsopermits a very high throughput iterative screening strategy: first,multiple beads are placed in 96-well microtiter plates; second,compounds are partially detached and transferred to assay plates; third,a metal binding assay identifies the active wells; fourth, thecorresponding beads are rearrayed singly into new microtiter plates;fifth, single active compounds are identified; and sixth, the structuresare decoded.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

General Methods Utilized in the Examples

NMR spectra were acquired at proton frequencies of 270 and 300 MHz,using CDCl₃ as solvent unless noted otherwise. ¹H chemical shifts arereported relative to Me₄Si (“TMS”; δ=0.00 ppm) or CHCl₃ (δ=7.26 ppm) asinternal standards; ³¹P chemical shifts are relative to external aqueous85% H₃PO₄ (δ=0.00 ppm); and ¹³C chemical shifts are relative to CHCl₃(6=77.00 ppm) or TMS (δ=0.00 ppm) as internal standards. Mass spectrawere obtained in electron impact ionization mode at 70 eV. Opticalrotations were measured at rt.

Example 1

Anhydrous sodium hypophosphite (prepared from sodium hypophosphitemonohydrate by azeotropic distillation with toluene in vacuo at 50° C.)(940 mg, 10.8 mmol) was suspended in 60 mL of dry CH₂Cl₂ and cooled to0° C., then triethylamine (2.50 mL, 18.9 mmol) and chlorotrimethylsilane(2.32 mL, 18.4 mmol) were added. After 5 min, compound 1 (500 mg, 1.54mmol) in 5 mL of dry CH₂Cl₂ was added. The mixture was stirred at rt for24 h before 1 N HCl (20 mL) was added. The reaction mixture wasextracted with CH₂Cl₂ (3×40 mL), and the combined organic phases weredried (MgSO₄). After concentration, the residue was dissolved in 6 mL ofdry CH₂Cl₂ and 0.6 mL of pyridine and cooled to 0° C., thentrimethylacetyl chloride (0.3 mL, 2.25 mmol) was added followed bybenzyl alcohol (0.21 mL, 1.8 mmol). The mixture was stirred at 0° C. tort for 2 h, then diluted with ether. The organic layer was washed with 1N HCl (10 mL), H₂O (10 mL), and brine (10 mL), dried (MgSO₄), andconcentrated. Flash chromatography over silica gel with CHCl₃-MeOH(30:1) as eluent gave racemic compound 2 (657 mg, 89%) as a colorlessoil: ¹H NMR (CDCl₃, 300 MHz) δ 7.22 (d, J_(H,P)=555 Hz, 0.6H), 7.19 (d,J_(H,P)=550 Hz, 0.4H), 7.39-7.34 (m, 15H), 5.15-4.95 (m, 6H), 2.98 (m,1H), 2.47-2.17 (m, 3H), 2.14-1.79 (m, 3H); ³¹P NMR (CDCl₃, 121 MHz) δ36.06, 35.08; ¹³C NMR (CDCl₃, 75 MHz) δ 173.46, 173.39, 172.13, 135.70,135.30, 128.73, 128.60, 128.58, 128.53, 128.46, 128.43, 128.38, 128.33,128.26, 128.20, 67.88, 67.79, 67.07, 66.99, 66.46, 38.05, 38.02, 31.25,30.02, 28.29, 28.13.

Example 2

To a solution of 2 (210 mg, 0.44 mmol) in 5 mL of dry THF was addedsodium hydride (15 mg, 60% dispersion in oil, 0.44 mmol) followed bycompound 1 (140 mg, 0.44 mmol) at 0° C., and the mixture was stirred atrt for 2 h before 1 N HCl was added. The reaction mixture was extractedwith CH₂Cl₂ (3×20 mL). The combined organic layers were dried (MgSO₄)and concentrated. Flash chromatography over silica gel with ethylacetate-hexanes (1:1) as eluent gave compound 3 (45 mg, 13%) as acolorless oil: ¹H NMR (CDCl₃, 300 MHz) δ 7.34-7.27 (m, 25H), 5.06-4.89(m, 10H), 2.83 (m, 2H), 2.33-2.14 (m, 6H), 2.00-1.60 (m, 6H); ³¹P NMR(CDCl₃, 121 MHz) δ −28.51, −28.90, −29.34; ¹³C NMR (CDCl₃, 75 MHz) δ173.80, 172.19, 136.27, 135.78, 135.50, 128.60, 128.56, 128.42, 128.33,128.28, 128.24, 128.19, 128.14, 66.91, 66.87, 66.80, 66.37, 66.10,66.02, 38.65, 38.51, 31.71, 30.32, 31.24, 30.67, 30.57, 30.04, 28.85,28.69.

Example 3

To a solution of 3 (42 mg, 0.52 mmol) in tert-butanol was added 30 mg of20% Pd(OH)₂/C (Aldrich, ≦50% H₂O), and the mixture was hydrogenatedunder 70 psi of H₂ for 24 h. The catalyst was removed by filtrationthrough celite, and the filtrate was concentrated. The residue wasdissolved in 5 mL of water and lyophilized to afford 17 mg (92%) of FN-6as a white solid: ¹H NMR (D₂O, 300 MHz) δ 2.77 (m, 2H), 2.44 (t, J=7.3Hz, 4H), 2.20 (dt, J=13.2, 11.3 Hz, 2H), 2.00-1.82 (m, 6H); ³¹P NMR(D₂O, 121 MHz) δ −33.12; ¹³C NMR (D₂O, 75 MHz) δ 179.69, 179.61, 178.61,39.89, 32.70, 32.28, 31.49, 29.68, 29.58, 29.52, 29.42.

Example 4

Compound 4 (130 mg, 0.33 mmol) was dissolved in 2 mL of dry CH₂Cl₂ and0.2 mL of pyridine and cooled to 0° C., then trimethylacetyl chloride(80 μL, 0.6 mmol) was added followed by (R)-(+)-1-phenyl-1-butanol (65mg, 0.44 mmol). The mixture was stirred at 0° C. to rt for 2 h, thendiluted with ether, and the reaction mixture was washed with 1N HCl (10mL), H₂O (10 mL), and brine (10 mL), dried (MgSO₄), and concentrated.Flash chromatography over silica gel with CHCl₃-MeOH (30:1) as eluentgave 5 (164 mg, 94%) as a mixture of four diastereoisomers. ¹H NMR(CDCl₃, 300 MHz) δ 7.45-7.28 (m, 15H), 7.26 (d, J_(H,P)=558 Hz, 0.24H),7.23 (d, J_(H,P)=554 Hz, 0.16H), 6.91 (d, J_(H,P)=552 Hz, 0.60H),5.37-5.24 (m, 1H), 5.17-5.03 (m, 4H), 3.05-2.68 (m, 1H), 2.56-1.60 (m,8H), 1.50-1.20 (m, 2H), 0.98-0.91 (m, 3H); ³¹P NMR (CDCl₃, 121 MHz) δ34.88, 34.10, 32.22, 31.32.

Example 5

To a solution of 5 (220 mg, 0.44 mmol) in 5 mL of dry THF was addedsodium hydride (15 mg, 60% dispersion in oil, 0.44 mmol) followed bycompound 1 (See Example 1; 140 mg, 0.44 mmol) at 0° C., and the mixturewas stirred at rt for 2 h. 1N HCl was added, and the mixture wasextracted with CH₂Cl₂ (3×20 mL). The combined organic phases were dried(MgSO₄) and concentrated. Flash chromatography over silica gel withCHCl₃-MeOH (30:1) as eluent gave a mixture of four isomers (110 mg,27%). ³¹P NMR showed four peaks: δ −29.91, −30.21, −30.37, and −30.66.Careful preparative TLC separation (ethyl acetate-hexanes 2:3) gavehomogeneous compounds 6a-d.

6a: R_(f) 0.60 (ethyl acetate-hexanes 1:1); [α]_(D) +12.1° (c 0.7,CHCl₃); ¹H NMR (CDCl₃, 300 MHz) δ 7.37-7.28 (m, 25H), 5.30 (dt, J=8.7,7.2 Hz, 1H), 5.20-4.97 (m, 8H), 2.91 (m, 1H), 2.56 (m, 1H), 2.39-2.22(m, 3H), 2.10-1.60 (m, 11H), 1.35-1.09 (m, 2H), 0.87 (t, J=7.2 Hz, 3H);³¹P NMR (CDCl₃, 121 MHz) δ −29.90; ¹³C NMR (CDCl₃, 75 MHz) δ 173.93,173.86, 173.78, 173.70, 172.23, 172.12, 140.98, 135.78, 135.55, 128.59,128.51, 128.46, 128.25, 128.23, 128.17, 126.55, 77.19, 77.10, 66.82,66.76, 66.31, 66.23, 40.45, 40.38, 38.67, 38.63, 38.59, 38.56, 32.18,31.30, 31.09, 30.93, 30.11, 29.66, 28.84, 28.69, 28.64, 28.48, 18.58,13.68.

6b: R_(f) 0.58 (ethyl acetate-hexanes 1:1); [α]_(D) +12.9° (c 0.35,CHCl₃); ¹H NMR (CDCl₃, 300 MHz) δ 7.37-7.28 (m, 25H), 5.30 (dt, J=9.0,6.6 Hz, 1H), 5.21-4.99 (m, 8H), 2.99 (m, 1H), 2.55 (m, 1H), 2.41-2.20(m, 3H), 2.10-1.62 (m, 11H), 1.40-1.20 (m, 2H), 0.86 (t, J=7.2 Hz, 3H);³¹P NMR (CDCl₃, 121 MHz) δ −30.32; ¹³C NMR (CDCl₃, 75 MHz) δ 173.94,173.90, 173.87, 173.81, 172.25, 172.15, 141.01, 135.82, 135.58, 135.52,128.62, 128.57, 128.54, 128.49, 128.32, 128.28, 128.26, 128.24, 128.22,128.21, 128.19, 126.58, 77.20, 77.16, 66.82, 66.79, 66.33, 66.25, 40.44,40.37, 38.70, 38.67, 38.43, 38.38, 32.25, 31.90, 31.34, 31.13, 31.00,30.75, 29.70, 28.81, 28.67, 28.53, 28.38, 18.55, 13.71.

6c: R_(f) 0.57 (ethyl acetate-hexanes 1:1); [α]_(D) 0° (c 0.42, CHCl₃);¹H NMR (CDCl₃, 300 MHz) δ 7.35-7.20 (m, 25H), 5.29 (dt, J=9.3, 6.9 Hz,1H), 5.17-4.94 (m, 8H), 2.91 (m, 1H), 2.45-2.25 (m, 3H), 2.20-1.20 (m,14H), 0.84 (t, J=7.5 Hz, 3H); ³¹P NMR (CDCl₃, 121 MHz) δ−30.11; ¹³C NMR(CDCl₃, 75 MHz) δ 173.84, 173.74, 173.70, 173.62, 172.25, 172.10,140.84, 135.80, 135.60, 135.49, 128.62, 128.53, 128.41, 128.32, 128.24,128.21, 126.68, 77.24, 77.15, 66.87, 66.79, 66.33, 66.25, 40.27, 40.21,38.86, 38.83, 38.46, 38.41, 31.95, 31.62, 31.30, 31.19, 30.80, 30.39,29.70, 28.64, 28.53, 28.48, 28.40, 18.63, 13.73.

6d: R_(f) 0.55 (ethyl acetate-hexanes 1:1); [α]_(D) −2.8° (c 0.42,CHCl₃); ¹H NMR (CDCl₃, 300 MHz) δ 7.41-7.20 (m, 25H), 5.31 (dt, J=9.3,6.9 Hz, 1H), 5.23-4.98 (m, 8H), 2.97 (m, 1H), 2.50-2.17 (m, 4H),2.15-1.82 (m, 6H), 1.80-1.50 (m, 5H), 1.45-1.20 (m, 2H), 0.85 (t, J=7.2Hz, 3H); ³¹P NMR (CDCl₃, 121 MHz) δ −30.57; ¹³C NMR (CDCl₃, 75 MHz) δ173.77, 173.70, 173.66, 173.59, 172.18, 172.06, 140.81, 135.77, 135.50,135.46, 128.59, 128.53, 128.51, 128.41, 128.30, 128.23, 128.19, 126.58,77.16, 77.05, 66.82, 66.31, 66.24, 40.29, 40.23, 38.79, 38.30, 38.24,31.96, 31.44, 31.34, 31.14, 30.81, 30.20, 28.95, 28.80, 28.69, 28.53,18.54, 13.69.

Example 6

The synthetic procedure outlined for the preparation of FN-6 (SeeExample 3) was utilized for the preparation of optically-purestereoisomers FN-6a-d.

FN-6a (yield 94%): white solid; ¹H NMR (D₂O, 300 MHz) δ 2.76 (m, 2H),2.45 (t, J=7.5 Hz, 4H), 2.14 (dt, J=12.9, 9.6 Hz, 2H), 2.00-1.89 (m,4H), 1.83 (dt, J=14.4, 4.2 Hz, 2H); ³¹P NMR (D₂O, 121 MHz) δ −37.63; ¹³CNMR (D₂O, 75 MHz) δ 179.95, 178.74, 40.16, 32.55 (d, J_(1,P)=90.23 Hz),32.38, 29.67 (d, J_(3,P)=11.48 Hz).

FN-6b (yield 96%): white solid; ¹H NMR (D₂O, 300 MHz) δ 2.76 (m, 2H),2.45 (t, J=7.8 Hz, 4H), 2.13 (dt, J=12.6, 10.5 Hz, 2H), 2.04-1.89 (m,4H), 1.83 (dt, J=15.0, 4.2 Hz, 2H); ³¹P NMR (D₂O, 121 MHz) δ −36.42; ¹³CNMR (D₂O, 75 MHz) δ 180.07, 178.76, 40.10, 32.67 (d, J_(1,P)=90.75 Hz),32.40, 29.57 (d, J_(3,P)=11.25 Hz).

FN-6c (yield 99%): white solid; ¹H NMR (D₂O, 300 MHz) δ 2.76 (m, 2H),2.45 (t, J=7.5 Hz, 4H), 2.09 (dt, J=12.6, 9.0 Hz, 2H), 2.04-1.88 (m,4H), 1.79 (dt, J=15.0, 4.2 Hz, 2H); ³¹P NMR (D₂O, 121 MHz) δ −37.96; ¹³CNMR (D₂O, 75 MHz) δ 179.88, 178.72, 40.13, 32.46 (d, J_(1,P)=90.75 Hz),32.36, 29.66 (d, J_(3,P)=12 Hz).

FN-6d (yield 96%): white solid; ¹H NMR (D₂O, 300 MHz) δ 2.76 (m, 2H),2.45 (t, J=7.5 Hz, 4H), 2.14 (dt, J=13.2, 9.6 Hz, 2H), 2.00-1.86 (m,4H), 1.85 (dt, J=14.7, 3.6 Hz, 2H); ³¹P NMR (D₂O, 121 MHz) δ −35.56; ¹³CNMR (D₂O, 75 MHz) δ 179.88, 178.72, 40.13, 32.46 (d, J_(1,P)=90.75 Hz),32.37, 29.66 (d, J_(3,P)=12 Hz).

Example 7

To a solution of 4 (400 mg, 1.23 mmol) in nitromethane was added 0.1 mLof Triton B (40% solution in methanol). The mixture was stirred at roomtemperature for 5 h. The nitromethane was removed under reducedpressure. Flash chromatography over silica gel with ethylacetate-hexanes (8:1 to 3:1) as eluent gave compounds 5 (200 mg, 42%)and 6 (150 mg, 17%) as colorless oil. 5: IR (film) ν 1730 cm⁻¹, ¹H NMR(CDCl₃, 300 MHz) δ 7.40-7.28 (m, 10H), 5.13 (s, 2H), 5.10 (s, 2H), 4.35(m, 2H), 2.45-2.15 (m, 4H), 2.08-1.82 (m, 2H); ¹³C NMR (CDCl₃, 75 MHz) δ173.52, 172.22, 135.67, 135.32, 128.66, 128.57, 128.51, 128.32, 128.28,73.01, 66.89, 66.48, 41.40, 31.41, 28.95, 26.94. 6: IR (film) ν 1731cm⁻¹¹H NMR (CDCl₃, 300 MHz) δ 7.39-7.20 (m, 20H), 5.15-5.02 (m, 8H),4.53 (m, 1H), 2.52-2.22 (m, 6H), 2.08-1.75 (m, 8H); ¹³C NMR (CDCl₃, 75MHz) δ 173.34, 172.10, 135.70, 135.37, 128.57, 128.40, 128.29, 128.27,84.35, 66.87, 66.44, 41.17, 41.06, 35.83, 35.62, 31.32, 27.55, 27.41.

Example 8

To a solution of compound 5 (58 mg, 0.15 mmol) and benzyl acrylate (25mg, 0.15 mmol) in 1 mL of dry CH₂Cl₂ was added a catalytic amount ofTriton B (40% solution in methanol). The mixture was stirred at roomtemperature for 4 h. The solvent was removed under reduced pressure andflash chromatography over silica gel with ethyl acetate-hexanes (5:1) aseluent gave compound 7 (75 mg, 91%). ¹H NMR (CDCl₃, 300 MHz) δ 7.40-7.25(m, 15H), 5.20-5.03 (m, 6H), 4.64-4.46 (m, 1H), 2.55-1.80 (m, 11H); ¹³CNMR (CDCl₃, 75 MHz) δ 173.63, 172.10, 171.34, 135.67, 135.49, 128.60,128.55, 128.41, 128.31, 128.27, 85.67, 85.03, 66.86, 66.67, 66.44,41.17, 35.41, 35.32, 31.35, 30.07, 29.96, 29.07, 28.36, 27.48, 26.64.

Example 9

To a solution of compound 8 (54 mg, 0.076 mmol) in 2 mL of dry CH₂Cl₂was added 18 uL of triethylamine, after being stirred at roomtemperature for 10 min, 80 mg of CATP was added. The resulting mixturewas stirred for additional 4 h. Then 10 mL of ether was added andfiltered through celite, and washed with ether. The filtrate wasconcentrated. Chromatography afforded 8 (30 mg, 58%): ¹H NMR (CDCl₃, 300MHz) δ 7.40-7.22 (m, 20H), 5.15-5.01 (m, 8H), 3.00-2.79 (m, 4H),2.50-2.30 (m, 6H), 2.00-1.78 (m, 4H); ¹³C NMR (CDCl₃, 75 MHz) δ 205.98,205.73, 174.20, 174.03, 172.45, 172.40, 135.78, 135.68, 128.53, 128.51,128.22, 128.16, 66.58, 66.33, 43.95, 39.27, 31.62, 31.59, 29.68, 26.66,26.59.

Example 10

To a solution of compound 7 (70 mg, 0.13 mmol) in 5 mL of dry CH₂Cl₂ wasadded 30 uL of triethylamine, after being stirred at room temperaturefor 10 min, 120 mg of CATP was added. The resulting mixture was stirredfor an additional 4 h, then 20 mL of ether was added and filteredthrough celite, and washed with ether. The filtrate was concentrated.Chromatography afforded 9 (40 mg, 60%). ¹H NMR (CDCl₃, 300 MHz) δ7.40-7.25 (m, 15H), 5.16-5.05 (m, 6H), 3.02-2.90 (m, 2H), 2.80-2.50 (m,5H), 2.36 (t, J=7.2 Hz, 2H), 2.00-1.89 (m, 2H); ¹³C NMR (CDCl₃, 75 MHz)δ 206.30, 174.20, 172.43, 172.36, 135.76, 135.70, 128.51, 128.49,128.20, 128.17, 128.15, 66.57, 66.47, 66.33, 43.92, 39.33, 37.06, 31.62,27.85, 26.65.

Example 11

To a solution of 9 (40 mg, 0.078 mmol) in tert-butanol was added 30 mgof 20% Pd(OH)₂/C (Aldrich, ≦50% H₂O), and the mixture was hydrogenatedunder 1 atm of H₂ for 4 h. The catalyst was removed by filtrationthrough celite, and the filtrate was concentrated. The residue wasdissolved in 5 mL of water and lyophilized to afford 18 mg (94%) of 1 asa syrup. 1: ¹H NMR (CDCl₃, 300 MHz) δ 3.03-2.74 (m, 5H), 2.59 (t, J=6.0Hz, 2H), 2.44 (t, J=7.2 Hz, 2H), 1.96-1.76 (m, 2H).

Example 12

To a solution of 8 (24 mg, 0.034 mmol) in tert-butanol was added 10 mgof 20% Pd(OH)₂/C (Aldrich, ≦50% H₂O), and the mixture was hydrogenatedunder 1 atm of H₂ for 4 h. The catalyst was removed by filtrationthrough celite, and the filtrate was concentrated. The residue wasdissolved in 5 mL of water and lyophilized to afford 10 mg (93%) of 2 asa syrup. 2: ¹H NMR (CDCl₃, 300 MHz) δ 3.03-2.72 (m, 6H), 2.45 (t, J=7.5Hz, 4H), 1.96-1.75 (m, 4H).

Example 13

To a suspension of dibenzyl L-glutamate tosylate (1 g, 2 mmol) in 20 mLof CH₂Cl₂ was added triphosgene (110 mg, 0.37 mmol). The mixture wascooled to −78° C., and Et₃N (0.53 mL, 4 mmol) was added. The reactionmixture was allowed to warm to room temperature and stirred for anadditional 2 h. The reaction mixture was diluted with 100 mL of EtOAcand washed with H₂O (3×30 mL), brine (30 mL) and dried over MgSO₄. Thesolvent was evaporated and the residue was dissolved in 5 mL of CH₂Cl₂,and excess hexane was added. The white solid 10 was collected (600 mg,88%). ¹H NMR (CDCl₃, 300 MHz) δ 7.40-7.25 (m, 20H), 5.17 (br s, 2H),5.13 (br s, 4H), 5.08 (s, 4H), 4.53 (dt, J=8.1, 4.8 Hz, 2H), 2.41 (m,4H), 2.19 (m, 2H), 1.97 (m, 2H); ¹³C NMR (CDCl₃, 75 MHz) δ 172.70,172.51, 156.53, 135.67, 135.12, 128.51, 128.46, 128.33, 128.14, 67.18,66.38, 52.47, 30.17, 27.84.

Example 14

To a solution of 10 (510 mg, 0.75 mmol) in tert-butanol was added 200 mgof 20% Pd(OH)₂/C (Aldrich, ≦50% H₂O), and the mixture was hydrogenatedunder 1 atm of H₂ for 24 h. The catalyst was removed by filtrationthrough celite, and the filtrate was concentrated. The residue wasdissolved in 15 mL of water and lyophilized to afford 235 mg (98%) ofFN11 as a white solid: [α]_(D) −16.4° (c 0.5, H2O); ¹H NMR (D₂O, 300MHz) δ 4.26 (br s, 2H), 2.51 (t, J=9.9 Hz, 4H), 2.18 (m, 2H), 1.98 (m,2H); ¹³C NMR (D₂O, 75 MHz) δ 178.59, 177.59, 160.55, 53.91, 31.36,27.51.

Example 15

To a suspension of dibenzyl L-glutamate tosylate (1 g, 2 mmol) in 20 mLof CH₂Cl₂ was added triphosgene (200 mg, 0.66 mmol). The mixture wascooled to −78° C., then Et₃N (0.60 mL, 4 mmol) was added. The reactionmixture was stirred at −78° C. for 2 h, then allowed to warm to roomtemperature and diluted with 100 mL of EtOAc and washed with H₂O (30mL), 1N HCl (30 mL) and brine (30 mL). The mixture was dried over MgSO₄.The solvent was evaporated and the residue was chromatographed on silicagel with hexanes/EtOAc (2:1) to give isocyanate (230 mg). The isocyanate(230 mg, 0.65 mmol) was dissolved in 5 mL of CH₂Cl₂, and dibenzylL-glutamate (213 mg, 0.65 mmol) was added at 0° C. The mixture wasstirred at room temperature for 24 h. Excess hexane was added. The whitesolid 11 was collected (440 mg, 99% from isocyanate). ¹H NMR (CDCl₃, 300MHz) δ 7.35-7.25 (m, 20H), 5.51 (d, J=7.8 Hz, 2H), 5.11 (s, 4H), 5.06(s, 4H), 4.52 (dt, J=7.8, 5.4 Hz, 2H), 2.41 (m, 4H), 2.19 (m, 2H), 1.98(m, 2H); ¹³C NMR (CDCl₃, 75 MHz) δ 172.79, 172.68, 156.78, 135.75,135.21, 128.57, 128.53, 128.38, 128.23, 128.19, 67.22, 66.42, 52.53,30.25, 27.88.

Example 16

The general procedure outlined in Example 14 was followed for thepreparation of FN16 from 11. FN16: ¹H NMR (D₂O, 300 MHz) δ 4.24 (m, 2H),2.45 (t, J=7.2 Hz, 4H), 2.15 (m, 2H), 1.95 (m, 2H); ¹³C NMR (D₂O, 75MHz) δ 178.48, 177.40, 160.34, 53.80, 31.34, 27.62.

Example 17

The general procedure outlined in Example 13 was followed for thepreparation of 12 from dibenzyl D-glutamate tosylate.

Example 18

The general procedure outlined in Example 14 was followed for thepreparation of FN13 from 12. FN13: [α]_(D) +17.1° (c 0.84, H₂O); ¹H NMR(D₂O, 300 MHz) and ¹³C NMR (D₂O, 75 MHz) were same as for FN11.

Example 19

To a suspension of dimethyl L-glutamate chloride (2.1 g, 10 mmol) in 40mL of CH₂Cl₂ was added triphosgene (1 g, 3.3 mmol). The mixture wascooled to −78° C., then Et₃N (3 mL, 20 mmol) was added. The reactionmixture was stirred at −78° C. for 0.5 h, then allowed to warm to roomtemperature and diluted with 100 mL of EtOAc and washed with H₂O (30mL), 1N HCl (30 mL) and brine (30 mL). The mixture was dried over MgSO₄.The solvent was evaporated and the residue was chromatographed on silicagel with hexanes/EtOAc (2:1) to give the corresponding isocyanate (870mg). The isocyanate (870 mg, 4.3 mmol) was dissolved in 10 mL of CH₂Cl₂,and dimethyl L-glutamate (752 mg, 4.3 mmol) was added at 0° C. Themixture was then stirred at room temperature for 24 h. Excess hexane wasadded, and the white solid FN15 was collected (1.50 g, 92% fromisocyanate). ¹H NMR (CDCl₃, 300 MHz) δ 5.47 (d, J=7.8 Hz, 2H), 4.50 (dt,J=8.1, 5.1 Hz, 2H), 3.75 (s, 6H), 3.67 (s, 6H), 2.42 (m, 4H), 2.17 (m,2H), 1.96 (m, 2H); ¹³C NMR (CDCl₃, 75 MHz) δ 173.47, 173.36, 156.71,52.45, 52.40, 51.79, 30.03, 27.92.

Example 20

To a solution of dimethyl L-glutamate (575 mg, 3.28 mmol) in 15 mL ofCH₂Cl₂ was added thiophosgene (187 mg, 1.64 mmol). The mixture wascooled to −78° C., then Et₃N (0.43 mL, 3.28 mmol) was added. Thereaction mixture was stirred at −78° C. for a short period, then allowedto warm to room temperature over 15 h. The mixture was then diluted with100 mL of EtOAc and washed with H₂O (30 mL), 1N HCl (30 mL) and brine(30 mL). The mixture was dried over MgSO₄. The solvent was evaporatedand the residue was chromatographed on silica gel with hexanes/EtOAc(3:1→1:1) to give FN18 (410 mg, 64%). ¹H NMR (CDCl₃, 300 MHz) δ 5.08 (d,J=7.8 Hz, 2H), 4.50 (m, 2H), 3.78 (s, 6H), 3.68 (s, 6H), 2.46 (m, 4H),2.25 (m, 2H), 2.12 (m, 2H); ¹³C NMR (CDCl₃, 75 MHz) δ 182.89, 173.47,172.89, 56.19, 52.57, 51.82, 29.87, 27.40.

Example 21 Antitumor Activity of FN11

Implanted xenografts were formed by s.c. inoculation with 2×10⁶ U-87glioblastoma multiforme cells mixed with 0.5 mg Matrigel. FN11 wasdissolved in PBS to a concentration of either 10 or 100 μM, andinjections (0.1 mL) were made once daily into the base of the s.c.implanted xenografts. The starting volumes of the tumors wereapproximately 250 mm³. The results from this protocol are depicted inFIG. 15.

Example 22 Antiangiogenesis Activity of FN11

Control tumors and tumors grown under the conditions described inExample 21 were harvested at the end of 7 days, fixed in formalin andembedded in paraffin for sectioning. Tissue was stainedimmunohistochemically for von Willebrand's factor (vWF) to obtain ameasure of vascularization. FIGS. 13 and 14 present the results of thisprotocol for the tumor treated with FN11 at 100 μM. FIG. 13 was taken atlow magnification (100×), whereas FIG. 14 was taken at highmagnification (400×). The Figures reveal a preponderance of avascularand low vascular areas, mostly small vessels, in the tumor treated withFN11 at 100 μM.

Example 23 Studies of Neuroprotective Properties of FN Compounds

The testing of neuroprotective effects of ligands acting at metabotropicglutamate receptors requires the use of specific models that allow tovisualize the action of these compounds. As it has been shown in severalpublications, protection against excitotoxicity (which may be induced byNMDA application) often requires the presence of glial cells, and cannotbe demonstrated in cultures containing only neuronal cells. It isbelieved that the neuroprotective action of group II mGluR agonistsresult form an action at mGluR3 or mGluR2 receptors located on glialcells, which induces the release of neurotrophic factors from theseglial cells.

We have tested the validity of these statements using three models ofNMDA toxicity in cultures of cortical neurons prepared from fetal mice.The first model (A) consists of using a culture containing neuronswithout glial cells. NMDA (75 μM) is applied for 10 min without or withthe tested compound. Then the culture medium is replaced and cells areleft for 24 hours to develop toxicity. The second model (B) involves theuse of mice cortical neurons which are seeded on a layer of confluentglial cells (mostly astrocytes) prepared from cerebral cortex of newbornmice. The incubations are identical as in model A. The third model (C)separates the neuronal and glial cultures. Glial cultures are treatedwith the tested neuroprotective compound in order to induce the releaseof protective factors into the medium. In parallel, cultures of corticalneurons (without glia) are treated for 10 min with NMDA (20 μM). ThenNMDA is washed out and the neurons are treated with the medium collectedfrom the treated glial cells. In all cases toxicity is assessed 24 hoursafter NMDA treatment by measurements of lactate dehydrogenase (LDH)activity accumulated in the medium during this period of time.

The comparison of the three models is shown in FIG. 29. As expected, inmodel A ABHxD-I was not able to protect against NMDA toxicity. Incontrast, when glial cells were present (model B) ABHxD-I produced asignificant protection, reducing by over 40% the toxic effect of NMDA.This suggested that, in fact, ABHxD-I may induce the release ofprotective factors from glial cells. This possibility was tested inmodel C. As shown in FIG. 29, when incubated separately with glialcells, ABHxD-I was as effective as in model B. It should be noted thatmedium from untreated glial cells was not neuroprotective. This confirmsthe hypothesis that the action of ABHxD is mediated through the releaseof neuroprotective factors from glial cells. More importantly, thisindicates that the neuroprotective effect of ABHxD-I does not depend onthe presence of the drug during the toxic event, but the drug may beeffective when added after the toxic stimulus.

The comparison of models B and C shows that both models reflect theindirect neuroprotective effect of ABHxD-I mediated through the actionof the drug at receptors located on glial cells. This stresses the roleof glial cells in mechanisms of brain injury and identifies these cellsas a target of possible therapeutic intervention. From a practical pointof view, for the purpose of testing compounds which act through thismechanism, model C appears superior to model B, as the variability ofmeasurements is reduced in model C. This is reflected by a two-folddecrease in the standard error for the calculated means. This differencecomes, most probably, from the nature of the LDH measurements. In modelC, LDH can be only released from dying neurons and therefore it directlyreflects the number of dead cells. In contrast, in model B, LDH levelsmay be affected by LDH leakage from glial cells. As the number of glialcells is large compared to neurons, even small leakage may result insignificant changes in LDH levels. It is known that glial cells are notsensitive to NMDA toxicity, hence such increases do not reflect NMDAtoxicity, but rather random variation which occurs in every livingsystem. Hence, in model B, LDH release from dying neurons is measured ona higher background of variable control levels, which decreases thesignal-to-noise ratio and increases the random variability ofmeasurements. In our studies we initially used model B (as seen in FIGS.30 and 31), but then switched to model C (FIGS. 32 and 33) whichprovided more reliable measurements.

Testing of FN Compounds

Initially, FN compounds were tested in model B. FIG. 30 shows theeffects of FN1-FN8 used at 300 μM concentrations. Only FN1 and FN6 showabout 30% of neuroprotection. Among FN6 enantiomers used at 100 μMconcentrations (FIG. 30), the 6/1 and 6/4 compounds appear moreeffective.

FN6 enantiomers were also tested in model C (FIG. 32). As in model B,the 6/1 and 6/4 compounds were more effective, the protection reachingabout 30%. The Guilford compound PMPA, which is similar to FN6 was lesseffective. We also tested several newer FN compounds in model C. Asshown in FIG. 33, among those compounds only FN13 showed someneuroprotective effect which amounted to about 20% of protection.

SUMMARY

The strongest effects—about 30%—are shown by FN1 and FN6. These effectsare less than the neuroprotection seen with ABHxD-I, but better than theaction of PMPA. The tested compounds may show greater efficacy in modelsof toxicity with a smaller necrotic component and a larger apoptoticcomponent.

Example 24

To a stirred solution of 3-aminopropionitrile fumarate (1.1 g, 8.6 mmol)in 30 mL DMF was added 1 (2.31 g, 6.85 mmol) followed by BOP (3.50 g,7.94 mmol). The reaction mixture was cooled to 0° C. with an ice bathand Et₃N (2.2 mL, 15.8 mmol) was added. After stirring overnight atambient temperature, the reaction mixture was poured into ice-cold waterand extracted with EtOAc. The combined organic layers were washedsuccessively with 1 N HCl, H₂O, saturated NaHCO₃ and brine. The organiclayer was dried over Na₂SO₄, filtered and concentrated. The residue waschromatographed on SiO₂ (EtOAc/hexane 1:4, gradient elution) to affordproduct 2 (1.60 g, 60.0%).

Example 25

To a stirred suspension of 2 (1.60 g, 4.11 mmol) and triphenylphosphine(2.82 g, 10.8 mmol) in ice-cold anhydrous CH₃CN (45 mL) were addeddiisopropyl azodicarboxylate (2.2 mL, 11.2 mmol) and, 2 min later,trimethylsilyl azide (1.6 mL, 11.8 mmol) over 5 min. The heterogeneousreaction mixture was allowed to warm to ambient temperature, thenstirred overnight. To the mixture cooled to 0° C. was added a solutionof NaNO₂ (0.31 g, 4.5 mmol) in H₂O (1.5 mL). After 30 min, a solution ofceric ammonium nitrate (2.5 g, 4.5 mmol) in H₂O (7.0 mL) was added andthe mixture was stirred for 20 min. The mixture was poured into coldwater and extracted with CH₂Cl₂. The combined organic layers were washedwith H₂O, dried over Na₂SO₄, and concentrated in vacuo. The residue waschromatographed to afford 3 (1.05 g, 61.7%). ¹H NMR (300 MHz, CDCl₃) δ7.35 (s, 5H), 5.71 (d, 1H, J=8.1 Hz), 5.17 (dd, 1H, J=7.5, 16.2 Hz),5.11 (s, 2H), 4.80-4.64 (m, 2H), 3.06 (t, 2H, J=6.9 Hz), 2.70-2.43 (m,2H), 2.37-2.30 (m, 2H); ¹³C NMR (75 MHz, CDCl₃) δ 172.275, 155.606,155.292, 135.374, 128.417, 128.183, 128.103, 115.905, 80.676, 66.473,43.268, 42.446, 29.774, 28.310, 27.976, 18.221.

Example 26

To a stirred solution of 3 (205 mg, 0.50 mmol) in dry CH₂Cl₂ (3.0 mL)was added CF₃COOH (1.5 mL). After being stirred at ambient temperaturefor 3 h, the reaction mixture was poured into CH₂Cl₂ (20 mL), washedsuccessively by saturated NaHCO₃ and H₂O. The organic layer was driedover Na₂SO₄ and concentrated to afford the crude amine 4 (145 mg,92.3%).

To a stirred solution of the above amine 4 (145 mg, 0.46 mmol) in CH₂Cl₂(5 mL) at −78° C. was added Et₃N (0.064 mL, 0.46 mmol) and triphosgene(22.9 mg 0.23 mmol in 0.50 mL CH₂Cl₂), allowed to warm to roomtemperature and stirred for an additional 2 h. Diluted with CH₂Cl₂ andwashed with H₂O, and dried by Na₂SO₄. Filtered, concentrated and columnchromatography afforded 5 (70 mg, 46.5%). ¹H NMR (300 MHz, CDCl₃) δ7.40-7.25 (m, 10H), 6.26 (d, 2H, J=8.1 Hz), 5.29-5.21 (m, 2H), 5.77,5.25 (4H of AA system, J=12.6 Hz), 4.76-4.60 (m, 4H), 3.01 (t, 4H, J=6.9Hz), 2.25-2.20 (m, 8H); ¹³C NMR (75 MHz, CDCl₃) δ 172.468, 156.722,156.187, 135.501, 128.657, 128.464, 128.337, 116.032, 66.721, 42.880,42.727, 29.988, 28.751, 18.372.

Example 27

To a solution of 5 (11 mg, 0.168 mmol) in tert-butanol (10 mL) was added11 mg of 20% Pd(OH)₂/C (Aldrich, ≦50% H₂O), and the mixture washydrogenated under 1 atm of H₂ for 1 h. The catalyst was removed byfiltration through celite, and the filtrate was concentrated. Theresidue was dissolved in 5 mL of water and lyophilized to afford ca. 8mg of 6. ¹H NMR (300 MHz, D₂O) δ 5.22 (t, 2H, J=7.2 Hz), 4.82-4.77(mostly blanketed by water peak, 4H), 3.25 (t, 4H, J=6.6 Hz), 2.49 (t,4H, J=6.9 Hz), 2.27 (dd, 4H, J=6.9, 14.1 Hz); ¹³C NMR (75 MHz, D₂O) δ177.190, 158.035, 156.751, 118.100, 43.632, 43.164, 30.024, 27.705,18.000.

Example 28

To a solution of 6 (8 mg) in CH₂Cl₂ (3 mL) was added DBU (0.1 mL). Thereaction mixture was stirred at ambient temperature for 2 h,concentrated in vacuo. The residue was dissolved in H₂O and placed on aDowex-80 column. The product was eluted with water, then lyophilized toafford the final product ZJ04 (4 mg). ¹H NMR (300 MHz, D₂O) δ 5.16 (dd,2H, J=6.0, 9.0 Hz), 2.52 (t, 4H, J=6.9 Hz), 2.38-2.29 (m, 4H); ¹³C NMR(75 MHz, D₂O) δ 177.096, 158.583, 158.369, 45.223, 29.937, 27.972.

Example 29

Tetrazole 10 was prepared from amide 9 using the procedure described inExample 25. ¹H NMR (300 MHz, CDCl₃) δ 7.39 (s, 5H), 5.33 (d, 1H, J=6.0Hz), 5.21, 5.15 (2H of AA system, J=12.3 Hz), 4.47 (t, 2H, J=6.9 Hz),4.41-4.49 (m, 1H), 3.05 (t, 2H, J=6.9 Hz), 3.00-2.93 (m, 2H), 2.60-2.54(m, 1H), 2.30-2.23 (m, 1H), 1.44 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ172.468, 156.722, 156.187, 135.501, 128.657, 128.464, 128.337, 116.032,66.721, 42.880, 42.727, 29.988, 28.751, 18.372.

Example 30

Compound 12 was prepared in two steps from tetrazole 10 using theprocedure described in Example 26. 12: ¹H NMR (300 MHz, CDCl₃) δ7.34-7.29 (m, 10H), 6.23 (d, 2H, J=8.1 Hz), 5.12 (t, 4H, J=12.9 Hz),4.59-4.48 (m, 2H), 4.47-4.36 (m, 4H), 2.95-2.89 (m, 8H), 2.56-2.47 (m,2H), 2.28-2.18 (m, 2H); ¹³C NMR (75 MHz, CDCl₃) δ 172.321, 157.651,154.924, 135.201, 128.617, 128.457, 128.009, 116.640, 67.289, 52.424,42.245, 29.266, 19.414, 18.305.

Example 31

Compound 13 was prepared from 12 using the procedure described inExample 27. 13: ¹H NMR (300 MHz, D₂O) 4.72 (t, 4H, J=6.3 Hz), 4.29 (dd,2H, J=4.8, 9.6 Hz), 3.19 (t, 4H, J=6.3 Hz), 3.10 (t, 4H, J=7.2 Hz),2.56-2.41 (m, 2H), 2.28-2.16 (m, 2H).

Example 32

Compound ZJ05 was prepared from 13 using the procedure described inExample 28. ZJ05: ¹H NMR (300 MHz, D₂O) δ 4.08 (dd, 2H, J=4.8, 9.0 Hz),3.00 (t, 4H, J=7.2 Hz), 2.32-2.21 (m, 2H), 2.11-2.02 (m, 2H).

Example 33

To a solution of L-aspartate dibenzylester 1 (518 mg, 1.65 mmol) indichloromethane (20 mL) triphosgene (74 mg, 0.25 mmol) was added. Thesolution was cooled to −78° C. and TEA (0.46 mL, 3.3 mmol) was addeddropwise. After the addition was complete, the solution was allowed towarm to rt and stirred for 3 h. The mixture was washed with 1 N HCl (2×5mL) and with brine, dried over anhydrous Na₂SO₄ and evaporated. Theresidue was dissolved in dichloromethane and an excess of hexanes wasadded. The white precipitate was collected by suction filtration (378mg, 0.58 mmol). The product 2 was purified by flash-chromatography onsilica gel (hexanes/EtOAc 2:1). ¹H NMR (CDCl₃) of 2: δ 2.85-3.08 (ABq,J=17 Hz, both part d with J=4.5 Hz, 4H), 4.81 (m, 2H), 5.06 (s, 4H),5.12 (s, 4H), 5.50 (d, J=8.1 Hz, 2H), 7.30 (m, 20H).

Compound 2 (300 mg, 0.46 mmol) was dissolved in a mixture ofethylacetate/t-BuOH 2:1 (18 mL), 20% Pd(OH)₂/C (120 mg) was added andthe mixture was hydrogenated at 1 atm overnight. The catalyst wasfiltered off and washed with methanol. After evaporation of the solventthe residue was dissolved in distilled water and lyophylized to give 120mg of PC103. Overall yield: 50%. ¹H NMR (CD₃OD) of PC103: δ 2.68-2.79(ABq, J=17 Hz, both part d with J=5.0, 5.5 Hz, 4H), 4.52 (t, J=5.0 Hz,2H). ¹³C NMR (CD₃OD) of PC103: δ 37.85, 50.64, 159.73, 174.32, 175.09.Anal. (C₉H₁₂N₂O₉.0.2H₂O.0.2 t-BuOH) C, H, N. mp: >142° C. [α]²⁰_(D)=+36.84 (c=0.19, MeOH).

Example 34

To a mixture of L-glutamate dibenzylester tosylate 3 (1.5 g, 3 mmol) indichloromethane (20 mL) triphosgene (300 mg, 0.99 mmol) was added. Themixture was cooled to −78° C. and TEA (0.9 mL, 6 mmol) was addeddropwise. The solution was stirred for 3 h at −78° C. The mixture wasallowed to warm to rt, then it was diluted with 20 mL ofdichloromethane, washed with 1N HCl (2×5 mL) and brine, dried overanhydrous Na₂SO₄ and evaporated. The crude isocyanate 4 was purified bymeans of a rapid flash-chromatography on silica gel (hexanes/EtOAc 4:1)and immediately used in the next step.

The isocyanate 4 (172 mg, 0.487 mmol) was dissolved in dichloromethane(10 mL) and cooled to 0° C. and a solution of L-aspartate dibenzylester1 (152 mg, 0.487 mmol) in dichloromethane (5 mL) was added with asyringe. The mixture was stirred at rt overnight. After evaporation ofthe solvent, product 5 was purified by flash-chromatography on silicagel (hexanes/EtOAc 2:1) (yield: 80%). ¹H NMR (CDCl₃) of 5: δ 1.98 (m,1H), 2.18 (m, 1H), 2.41 (m, 2H), 2.84 (dd, J=17.5, 4.5 Hz, 1H), 3.06(dd, J=17.5, 4.5 Hz, 1H), 4.54 (m, 1H), 4.79 (m, 1H), 5.10 (m, 9H), 5.45(d, J=8.5 Hz, 1H), 7.30 (m, 20H).

Compound 5 (260 mg, 0.39 mmol) was dissolved in a mixture ofethylacetate/t-BuOH 2:1 (15 mL), 20% Pd(OH)₂/C (102 mg) was added andthe mixture was hydrogenated at 1 atm overnight. The catalyst wasfiltered off and washed with methanol. After evaporation of the solventthe residue was dissolved in distilled water and lyophylized to give 102mg of PC107 (yield: 85%). ¹H NMR (D₂O) of PC107: δ 1.97 (m, 1H), 2.16(m, 1H), 2.51 (t, J=7 Hz, 2H), 2.91 (m, 2H), 4.25 (dd, J=5, 9 Hz, 1H),4.59 (t, J=5.5 Hz, 1H). ¹³C NMR (CD₃OD) of PC107: 28.91, 31.08, 37.80,50.59, 53.51, 159.95, 174.34, 175.07, 175.88, 176.48. Anal. (C₁₀H₁₄N₂O₉)C, H, N. mp: >146° C. [α]²⁰ _(D)=+29.33 (c=0.3, MeOH).

Example 35

To a mixture of L-glutamate dibenzylester tosylate 3 (0.5 g, 1 mmol) indichloromethane (5 mL) triphosgene (98 mg, 0.33 mmol) was added. Themixture was cooled to −78° C. and TEA (0.28 mL, 2 mmol) was addeddropwise. The solution was stirred for 3 h at −78° C. A solution ofglycine benzylester (170 mg, 1 mmol) in 3 mL of dichloromethane wasadded with a syringe and the mixture was stirred at rt overnight. Themixture was washed with 1 N HCl (2×5 mL) and brine, dried over anhydrousNa₂SO₄ and evaporated. The crude product 6 was purified byflash-chromatography on silica gel (hexanes/EtOAc 4:1-2:1) to give 150mg of a colourless oil (yield: 30%). ¹H NMR (CDCl₃) of 6: δ 1.99 (m,1H), 2.18 (m, 1H), 2.43 (m, 2H), 3.98 (m, 2H), 4.58 (m, 1H), 5.07 (s,2H), 5.13 (s, 4H), 5.51 (bs, 1H), 5.73 (bs, 1H), 7.31 (m, 15H).

Compound 6 (150 mg, 0.29 mmol) was dissolved in 10 mL of t-BuOH. 20%Pd(OH)₂/C (76 mg) was added and the mixture was hydrogenated at 1 atmovernight. The catalyst was filtered off and washed with methanol. Afterevaporation of the solvent the residue was dissolved in distilled waterand lyophylized to give 66 mg of PC108 (yield: 92%). ¹H NMR (D₂O) ofPC108: δ 1.98 (m, 1H), 2.17 (m, 1H), 2.51 (t, J=7 Hz, 2H), 3.90 (s, 2H),4.27 (dd, J=5.5, 8.5 Hz, 1H). ¹³C NMR (CD₃OD) of PC108: δ 28.86, 31.09,42.61, 53.59, 160.43, 174.18, 175.84, 176.56. Anal. (C₈H₁₂N₂O₇) C, H, N.mp: >135° C. [α]²⁰ _(D)=+2.8 (c=0.25, MeOH).

Example 36

To a mixture of L-glutamate dibenzylester tosylate 3 (0.5 g, 1 mmol) indichloromethane (5 mL) triphosgene (98 mg, 0.33 mmol) was added. Themixture was cooled to −78° C. and TEA (0.28 mL, 2 mmol) was addeddropwise. The solution was stirred for 3 h at −78° C. t-Butylamine (0.21mL, 2 mmol) was added with a syringe and the mixture was stirred at rtovernight. The mixture was washed with 1 N HCl (2×5 mL) and with brine,dried over anhydrous Na₂SO₄ and evaporated. The crude product 7 waspurified by flash-chromatography on silica gel (hexanes/EtOAc 2:1) togive 318 mg of a colorless oil (yield: 74%). ¹H NMR (CDCl₃) of 7: δ 1.30(s, 9H), 1.99 (m, 1H), 2.18 (m, 1H), 2.43 (m, 2H), 3.98 (m, 2H), 4.26(bs, 1H), 4.51 (m, 1H), 4.79 (bs, 1H), 5.08 (s, 2H), 5.15 (d, J=2 Hz,2H), 7.33 (m, 10H).

Compound 7 (266 mg, 0.62 mmol) was dissolved in 15 mL of t-BuOH, 20%Pd(OH)₂/C (164 mg) was added and the mixture was hydrogenated at 1 atmovernight. The catalyst was filtered off and washed with methanol. Afterevaporation of the solvent the residue was dissolved in distilled waterand lyophylized to give 149 mg of PC113 (yield: 95%). ¹H NMR (CD₃OD) ofPC113: δ 1.21 (s, 9H), 1.79 (m, 1H), 2.03 (m, 1H), 2.29 (m, 2H), 4.18(dd, J=5, 8.5 Hz, 1H). ¹³C NMR (CD₃OD) of PC113: δ 29.08, 29.71, 31.19,50.86, 53.21, 159.77, 176.30, 176.60. Anal. (C₁₀H₁₈N₂O₅.0.1H₂O) C, H, N.mp: 108-110° C. [α]²⁰ _(D)=+8.08 (c=0.52, MeOH).

Example 37

A solution of L-cysteine (1 g, 8.25 mmol) in 7.85 mL of 2 N HCl and 1 mLof t-BuOH was refluxed for 24 h. After evaporation of the solvent, thecrude material was dissolved in benzene (10 mL) and benzyl alcohol (4.7mL, 41.25 mmol). p-Toluenesulfonic acid (1.66 g, 8.75 mmol) was addedand the mixture was refluxed overnight with a Dean-Stark to collect thewater. The solution was cooled and a white solid precipitated, which wasfiltered, washed several times with ethyl ether and dried (yield: 55%).¹H NMR (D₂O) of 8: δ 1.28 (s, 9H); 2.38 (s, 3H); 3.15 (m, 2H); 4.43 (t,J=5 Hz, 1H); 5.32 (d, J=2.5 Hz, 2H); 7.35 (d, J=8 Hz, 2H); 7.46 (m, 5H);7.68 (d, J=6.5 Hz, 2H).

To a mixture of L-glutamate dibenzylester tosylate 3 (0.5 g, 1 mmol) indichloromethane (5 mL), triphosgene (98 mg, 0.33 mmol) was added. Themixture was cooled to −78° C. and TEA (0.28 mL, 2 mmol) was addeddropwise. After stirring for 2.5 h at −78° C., a solution of 8 (267 mg,1 mmol) in 5 mL of dichloromethane was added with a syringe. The mixturewas stirred at rt overnight. The mixture was washed with 1 N HCl (2×5mL) and brine, dried over anhydrous Na₂SO₄ and evaporated. The crudeproduct 9 was purified by flash-chromatography on silica gel(hexanes/EtOAc 2:1) to give 350 mg of a yellow oil (yield: 56%). ¹H NMR(CDCl₃) of 9: δ 1.26 (s, 9H); 2.01 (m, 1H); 2.19 (m, 1H); 2.41 (m, 2H);2.98 (m, 2H); 4.56 (m, 1H); 4.78 (m, 1H); 5.09 (s, 2H); 5.10 (s, 2H);5.18 (d, J=8 Hz, 1H); 5.29 (d, J=8 Hz, 1H); 7.34 (m, 15H).

Compound 9 (300 mg, 0.48 mmol) was dissolved in 10 mL of t-BuOH, 20%Pd(OH)₂/C (125 mg) was added and the mixture was hydrogenated at 1 atmfor 24 h. The catalyst was filtered off and washed with methanol. Afterevaporation of the solvent 123 mg of a yellow solid 10 was obtained.(yield: 73%). A small sample was dissolved in water, washed with ethylacetate and lyophilized to give a pure compound 10 (PC120). ¹H NMR(CD₃OD) of PC 120: δ 1.29 (s, 9H); 1.88 (m, 1H); 2.10 (m, 1H); 2.38 (m,2H); 2.94 (d, J=5.5 Hz, 2H); 4.29 (m, 1H); 4.49 (m, 1H). ¹³C NMR (CD₃OD)of PC 120: δ 29.02, 31.09, 31.26, 31.83, 43.07, 53.50, 54.33, 159.75,174.61, 175.80, 176.50. Anal. (C₁₃H₂₂N₂O₇S.0.5H₂O) C, H, N. mp: >145° C.dec. [α]²⁰ _(D)=+4.0 (c=0.1, MeOH).

Compound 10 (85 mg, 0.24 mmol) was dissolved in 2 mL of trifluoroaceticacid and cooled to 0° C. One drop of anisole and Hg(OAc)₂ (80 mg, 0.24mmol) were added, and the solution was stirred at rt for 2 h. Afterevaporation of the solvent, the solid residue was washed with ethylether and dried. The fine powder obtained was dissolved in MeOH, and H₂Swas bubbled into the solution for 5 minutes. The black precipitate wasfiltered through Celite and the solution was evaporated to give 70 mg ofPC 118 as a white solid (yield: 99%). ¹H NMR (CD₃OD) of PC118: δ 1.80(m, 1H); 2.05 (m, 1H); 2.31 (m, 2H); 2.82 (d, J=4.5 Hz, 2H); 4.21 (dd,J=5, 8.5 Hz, 1H); 4.45 (t, J=4.5 Hz, 1H). ¹³C NMR (CD₃OD) of PC118: δ27.84, 28.83, 31.10, 53.55, 55.89, 159.83, 173.92, 175.93, 176.50. Anal.(C₉H₁₄N₂O₇S.0.3H₂O) C, H, N. mp: >135° C. [α]²⁰ _(D)=+14.2 (c=0.12,MeOH).

Example 38 Design of Remarkably Simple, Yet Potent Inhibitors ofGlutamate Carboxypeptidase II (NAALADase)

The overactivation of glutamate receptors has been implicated inneuronal loss in acute conditions such as head injury, stroke, andprolonged epileptic seizures, as well as in chronic neurodegenerativediseases, including Alzheimer's disease, Huntington's disease,Parkinson's disease, amyotrophic lateral sclerosis, and AIDS dementia.The susceptibility of neurons to glutamate-induced death may result fromcellular energy deficits, increased glutamate release, decreasedglutamate uptake by neurons or glia, or changes in glutamate receptorproperties or expression patterns. It has been hypothesized that theabundant brain dipeptide N-acetylaspartylglutamate (NAAG) may contributeto neurodegeneration through its breakdown to glutamate by the action ofglutamate carboxypeptidase II (GCPII; also known as N-acetylatedalpha-linked acidic dipeptidase, NAALADase, or NAAG peptidase).Previously we have shown that4,4′-phosphinicobis(butane-1,3-dicarboxylic acid) is able to act as amimic of NAAG as it possesses fairly good selectivity for the mGluR3subtype of metabotropic receptors. Additionally, and more importantly,it was shown to act as a nM potency inhibitor of GCPII. Because of thepossibility that GCPII inhibitors may provide a new class of therapeuticagents for the treatment of stroke and related diseases, we have carriedout additional SAR work in this area starting from our lead compound.Most notably, we now show that certain simple ureas, AA-C(O)-AA′,prepared from readily available amino acids (AA), are able to act as nMpotency inhibitors of GCPII. We also demonstrate that several of theseureas dose-dependently protect neuronal cells from excitatory amino acidtoxicity. The present discovery thus opens up a new avenue to therational design of GCPII inhibitors that may prove valuable asclinically effective neuroprotective agents.

The amino acid glutamate is present in high concentrations in themammalian brain, and it acts as the major excitatory neurotransmitter inthe CNS. Through its actions on both ionotropic and metabotropicreceptors, glutamate plays an important role in a variety ofphysiological functions including learning, memory, and developmentalplasticity. Excessive activation of glutamate receptors or disturbancesin the cellular mechanisms that protect against the adverse consequencesof physiological glutamate receptor activation have been implicated inthe pathogenesis of a host of neurological disorders. These disordersinclude epilepsy, ischemia, central nervous system trauma, neuropathicpain, and chronic neurodegenerative diseases. Although several drugsdesigned to attenuate the pathological consequences of excessiveglutamate activation have been shown to reduce injury in experimentalmodels of cerebral ischemia, so far none of these compounds has provento be effective in the clinical treatment of stroke.¹

N-acetyl-L-aspartyl-L-glutamate (NAAG) is a peptide neurotransmitterthat is widely distributed in the mammalian nervous system.² NAAG isreleased from neurons after depolarization in a calcium-dependentmanner,³ and it is both an agonist at group II metabotropic glutamatereceptors⁴ and a mixed agonist/antagonist at the N-methyl-D-aspartate(NMDA) receptor.⁵ NAAG is hydrolyzed by the neuropeptidase glutamatecarboxypeptidase II (GCPII; also known as N-acetylated alpha-linkedacidic dipeptidase, NAALADase, or NAAG peptidase) to liberateN-acetylaspartate and glutamate both in vitro and in vivo.⁶ The role ofGCPII is thus thought to be two-fold: (1) to terminate theneurotransmitter activity of NAAG; and (2) to liberate glutamate whichis then able to act at the various glutamate receptor subtypes.Alterations in the levels of GCPII and NAAG have been observed indisorders that are linked to abnormalities in glutamatergicneurotransmission.⁷

As a consequence of these findings, it has been hypothesized that theinhibition of GCPII might provide an effective strategy for achievingneuroprotection in cases of cerebral ischemia by increasing the levelsof NAAG while decreasing the levels of glutamate. In fact, recent workby Slusher et al. led to the demonstration that the GCPII inhibitor2-PMPA provides significant protection against injury in rats aftertransient middle cerebral artery occlusion (MCAO).⁸ Furthermore, in therat MCAO model, 2-PMPA decreased glutamate levels while increasing NAAGlevels, as would be predicted for a compound working as a GCPIIinhibitor. As a therapeutic target, GCPII inhibition has been suggestedto have potential benefits over receptor-based strategies, as itrepresents an upstream mechanism of glutamate regulation that couldreduce transmission at a number of glutamatergic receptors rather thaninhibiting a single receptor subtype.^(8,9) Equally important, NAAG iscolocalized in neurons with small amine transmitters including GABA anddopamine, and it has been shown to act on presynaptic receptors toregulate the synaptic release of these transmitters.¹⁰

In our previous work,¹¹ starting from NAAG, we designed a dually actingligand, 4,4′-phosphinicobis(butane-1,3-dicarboxylic acid) (PBBDA), whichacts both as an mGluR3 selective agonist (˜30 μM) and as a potentinhibitor of GCPII (21.7±2.1 nM). From this novel lead compound, we nowchose to investigate the activity of structures comprised of two aminoacids joined through their NH₂ groups by a urea linkage (FIG. 1). Weenvisaged that the urea group would serve as a suitable replacement forthe central CH₂P(O)(OH)CH₂ present in the lead structure. The impetus topursue this chemistry was driven largely by the ease of synthesizingsuch structures, thereby facilitating further SAR analysis.

The compounds that have been prepared are shown in Table 1. Forcomparison purposes, we also provide published data^(6a) for somerelated dipeptides structures. First, we explored the activity ofGlu-C(O)-Glu, where Glu's are of S-configuration. In general, thesesymmetrical ureas were prepared (Scheme 1) by reacting the appropriateamino acid benzyl ester with triphosgene/Et₃N at −78° C., followed bywarming to rt. After purification by column chromatography orrecrystallization, the intermediate tetraester was transformed to itsfree acid by hydrogenolysis. All new compounds were assayed for theirability to inhibit rat GCPII stably expressed in Chinese Hamster Ovary(CHO) cells using conditions identical to those reported previously. Thereadily synthesized compound (S)Glu-C(O)—(S)Glu was quite active, withan IC₅₀ value of 47 nM against expressed rat GCPII. Thus, this ligand isonly two-fold less potent than our lead phosphinate. Glu-C(O)-Glu madefrom R-Glu gave only 67% inhibition when tested at 100 μM, while(R)-Glu-CO—(S)-Glu gave 97% inhibition at the same concentration, thusdemonstrating the specificity of the enzyme for S-configured aminoacids. The corresponding dipeptide (S)-Glu-(S)-Glu has been reported tohave some inhibitory activity toward GCPII, but it is 16-fold lesspotent with an IC₅₀ of 0.75 μM.^(6a,12) Interestingly, when we examined(S)-Asp-C(O)—(S)-Asp, this compound was surprisingly inactive, with anIC₅₀ of 3.8 μM. The corresponding dipeptide (S)-Asp-(S)-Asp has areported IC₅₀ of 58 μM.^(6a) Next, we examined the activity of(S)-Asp-C(O)—(S)-Glu, and found this compound to be comparable inactivity (IC₅₀=46 nM) to (S)-Glu-C(O)—(S)-Glu. Thus, the presence of asingle fragment having a three-carbon spacer between two of the carboxylgroups appears to be essential for high inhibitory potency. Thepossibility to replace the urea linker by a larger spacer group, namelyan oxalamide, was explored. This particular analog,(S)-Glu-C(O)C(O)—(S)-Glu, proved to be inactive. Lastly, we examined theability to replace one of the Glu fragments by other amino acids, oreven a simple amine. The preparation of these unsymmetrical ureas wasbrought about by first treating the tosylate salt of dibenzyl glutamatewith triphosgene/Et₃N at −78° C. followed by addition of the secondamine component and warming to room temperature. Deprotection was theneffected through catalytic hydrogenation as well as the use ofTFA/Hg(OAc)₂/anisole followed by H₂S in the case of cleavage of the t-Bugroup from Cys (Scheme 2).

As shown in Table 1, the urea derived from t-butylamine+(S)-Glu provedinactive, as did Gly-C(O)—(S)-Glu. In light of the ability of certainsulfur containing ligands to act as potent peptidase inhibitors (e.g.,captopril for angiotensin converting enzyme) through the ability of thesulfur atom to coordinate with a zinc atom present in the active site,we felt it would be valuable to explore the activity of(S)-Cys-C(O)—(S)-Glu. Remarkably, this tricarboxylic acid proved to benearly as potent as (S)-Glu-C(O)—(S)-Glu. Even the t-butylthiocontaining precursor molecule (t-Bu)Cys-CO—(S)-Glu proved to be activewith a K_(i) of about 100 nM. Note, however, that the related urea(S)-Cys-C(O)—(S)-Cys was inactive when tested at 1 μM. Thus the presentSAR reveals that a urea containing at least one glutamate residue plus asecond residue bearing a carboxyl group in addition to another group (SRor CO₂H) represents the minimum requirement to achieve effective GCPIIinhibition.

TABLE 1 Inhibitory activity of dipeptides and ureas against expressedrat GCPII. Compound IC₅₀ [HO₂C(CH₂)₂CH(CO₂H)CH₂]₂P(O)(OH) 26 nM (ref.11) (S)-Glu-(S)-Glu 0.75 μM (ref. 6a) (S)-Glu-C(O)—(S)Glu 47 ± 4.5 nM(R)-Glu-C(O)—(R)-Glu 67% inhibition at 100 μM (R)-Glu-C(O)—(S)-Glu 97%inhibition at 100 μM (S)-Glu-C(O)—C(O)—(S)-Glu 9% inhibition at 1 μM(S)-Asp-(S)-Asp 42% inhibition at 100 μM (S)-Asp-C(O)—(S)-Asp 3.8 μM(S)-Asp-(S)-Glu 2.4 μM (ref. 6a) (S)-Asp-C(O)—(S)-Glu 46.1 ± 1.4 nMt-BuNHC(O)—(S)-Glu 10% inhibition at 1 μM Gly-C(O)—(S)-Glu 46%inhibition at 1 μM (S)-Cys-C(O)—(S)-Cys Inactive at 1 μM(t-Bu)Cys-C(O)—(S)-Glu 100.9 ± 19.3 nM (S)-Cys-C(O)—(S)-Glu 72.4 ± 6.5nM

Moreover, as shown in FIG. 34, we have investigated the action of someof these novel ureas for their ability to block NMDA toxicity in vitro.This was done using a published protocol in which cultures of corticalglial cells are first treated with the test compound in order to inducethe release of protective factors into the medium.¹³ In parallel,cultures of cortical neurons (without glia) are exposed for 10 min tothe excitotoxic action of NMDA (20 EM). The NMDA is removed by washing,and the neurons are treated with the medium collected from the treatedglial cells. Toxicity is then assessed 24 hours after NMDA treatment bythe measurement of lactate dehydrogenase (LDH) activity, which serves asa marker for dying cells. In this test system 1 μM PMPA afforded onlyabout 25% neuroprotection. This result is consistent with the resultsobtained in other in vitro models where PMPA produced only a modestneuroprotection (16%) against NMDA-induced cell death.¹⁴ Among thepotent GCPII inhibitors tested, three compounds[HO₂C(CH₂)₂CH(CO₂H)CH₂]₂P(O)(OH), (S)-Glu-C(O)—(S)Glu, and(S)-Asp-C(O)—(S)Glu produced at 1 μM a similarly modest neuroprotectionranging from 21% to 31%. In contrast, the two Cys-containing compounds,(t-Bu)Cys-C(O)—(S)-Glu and (S)-Cys-C(O)—(S)-Glu, produced 69% and 50%neuroprotection, respectively. While the exact mechanism of thisneuroprotective effect cannot be explained based entirely upon GCPIIinhibition, these exciting results warrant further study in other modelsof in vitro toxicity, including the assessment of the compounds' effectsin vivo.

In conclusion, this Example reveals the ability of some remarkablysimple compounds to act as potent inhibitors of GCPII, thus offering anew avenue in the rational design of GCPII inhibitors that may lead toeffective neuroprotective agents. It is likely that the appendage ofother functionality, particularly hydrophobic groups, may lead tofurther improvements in potency through interaction with accessoryhydrophobic pockets. Moreover, because of the possibility to employGCPII inhibitors in stroke therapy, it will be essential to exploreprodrugs, or analogs containing carboxylic acid isosteres so as tofacilitate blood brain barrier penetration. Lastly, we note that thevalue of the urea motif in creating potent enzyme inhibitors has alsobeen recognized in the design of effective HIV protease inhibitors. 15

REFERENCES & NOTES FOR EXAMPLE 38

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INCORPORATION BY REFERENCE

All of the patents and publications cited herein are hereby incorporatedby reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of inhibiting NAALADase in a mammal, comprising the step ofadministering to a mammal an inhibitory effective amount of a compoundof the formula:

wherein Bn is benzyl; R₂ is selected from the group consisting ofhydrogen, alkyl, and amino; and wherein the stereochemical configurationat any stereocenter of the compound is R or S or a mixture thereof.
 2. Amethod of agonizing a metabotropic glutamate receptor in a mammal,comprising the step of administering to a mammal an agonizing effectiveamount of a compound of the formula:

wherein Bn is benzyl; R₂ is selected from the group consisting ofhydrogen, alkyl, and amino; and wherein the stereochemical configurationat any stereocenter of the compound is R or S or a mixture thereof.
 3. Amethod of antagonizing a metabotropic glutamate receptor in a mammal,comprising the step of administering to a mammal an antagonizingeffective amount of a compound of the formula:

wherein Bn is benzyl; R₂ is selected from the group consisting ofhydrogen, alkyl, and amino; and wherein the stereochemical configurationat any stereocenter of the compound is R or S or a mixture thereof.
 4. Amethod of agonizing a single subtype of metabotropic glutamate receptorin a mammal, comprising the step of administering to a mammal anagonizing effective amount of a compound of the formula:

wherein Bn is benzyl; R₂ is selected from the group consisting ofhydrogen, alkyl, and amino; and wherein the stereochemical configurationat any stereocenter of the compound is R or S or a mixture thereof.
 5. Amethod of antagonizing a single subtype of metabotropic glutamatereceptor in a mammal, comprising the step of administering to a mammalan antagonizing effective amount of a compound of the formula:

wherein Bn is benzyl; R₂ is selected from the group consisting ofhydrogen, alkyl, and amino; and wherein the stereochemical configurationat any stereocenter of the compound is R or S or a mixture thereof.
 6. Amethod of treating a neurological disorder selected from the groupconsisting of epilepsy, ischaemia, central nervous system trauma,neuropathic pain, and chronic neurodegenerative diseases, which methodcomprises the step of administering to a mammal an antagonizingeffective amount of a compound of formula 1:

wherein X is selected from the group consisting of —C(O)—, —C(S)—,—S(O)₂—, —C(R)(OR)—, and —C(R)(SR)—; Y is selected, independently foreach occurrence, from the group consisting of (CR₂)_(n), (NR)_(n), and abond; Z is selected, independently for each occurrence, from the groupconsisting of C(R), C(NR₂), and C(NHacyl); W is selected, independentlyfor each occurrence, from the group consisting of (CR₂)_(m), (NR)_(m),and a bond; G is selected, independently for each occurrence, from thegroup consisting of H, —COOH, —SO₃H, —P(O)(OH)₂, —SR, and2-R-tetrazol-5-yl; R is selected, independently for each occurrence,from the group consisting of H, alkyl, heteroalkyl, aryl, heteroaryl,and aralkyl; and also including a negative charge for instances of Rbonded to a heteroatom; m and n are integers selected, independently foreach occurrence, from the range 0 to 3 inclusive.
 7. A method oftreating a neurological disorder selected from the group consisting ofepilepsy, ischaemia, central nervous system trauma, neuropathic pain,and chronic neurodegenerative diseases, which method comprises the stepof administering to a mammal an antagonizing effective amount of acompound of the formula 2:

wherein X is selected from the group consisting of —C(O)—, —C(S)—,—P(O)(OR)—, —S(O)₂—, —C(R)(OR)—, and —C(R)(SR)—; Y is selected,independently for each occurrence, from the group consisting of(CR₂)_(n), (NR)_(n), and a bond; G is selected, independently for eachoccurrence, from the group consisting of H, —COOH, —SO₃H, —P(O)(OH)₂,and 2-R-tetrazol-5-yl; R is selected, independently for each occurrence,from the group consisting of H, alkyl, heteroalkyl, aryl, heteroaryl,and aralkyl; and also including a negative charge for instances of Rbonded to a heteroatom; n is an integer selected, independently for eachoccurrence, from the range 0 to 3 inclusive.
 8. A method of treating aneurological disorder selected from the group consisting of epilepsy,ischaemia, central nervous system trauma, neuropathic pain, and chronicneurodegenerative diseases, which method comprises the step ofadministering to a mammal an antagonizing effective amount of a compoundof the formula:

wherein Bn is benzyl; R₂ is selected from the group consisting ofhydrogen, alkyl, and amino; and wherein the stereochemical configurationat any stereocenter of the compound is R or S or a mixture thereof. 9.The method of any one of claims 6-8, wherein the neurological disorderis epileptic seizure.
 10. The method of any one of claims 6-8, whereinthe neurological disorder is schizophrenia.
 11. The method of any one ofclaims 6-8, wherein the neurological disorder is amyotrophic lateralsclerosis.